Recombinant Bovine Transmembrane protein 164 (TMEM164)

What is the basic structure of TMEM164?

TMEM164 is characterized by a six transmembrane (6TM) helical core structure. According to AlphaFold2 modeling, it contains a conserved Cys/His pair (specifically C123/H181 in human TMEM164) that forms the putative catalytic dyad. The protein features an internal cavity with predicted portals to the lipid bilayer and the cytosol positioned adjacent to this catalytic dyad. This structural arrangement is consistent with its function as a membrane-embedded enzyme .

What is the primary function of TMEM164?

TMEM164 functions as a cysteine-dependent acyltransferase that specifically generates C20:4 ether phospholipids (ePLs), particularly C20:4 ether phosphatidylethanolamines (ePEs). Unlike other acyltransferases, TMEM164 uses C20:4-phosphatidylcholine (C20:4-PC) as an acyl chain donor rather than C20:4-CoA to transfer arachidonic acid to lyso-ether phospholipids (lyso-ePLs). This enzymatic activity contributes to the maintenance of polyunsaturated fatty acid (PUFA)-containing ether lipids in cellular membranes .

How does TMEM164 relate to other known acyltransferases?

While TMEM164 performs acyltransferase functions similar to those in the MBOAT family (such as LPCAT3 and MBOAT7), it shares no discernible sequence motifs or predicted domain homology with these enzymes. Instead, structural predictions suggest TMEM164 belongs to a distinct transmembrane protein family that includes bacterial YwaF/YpiA proteins and the human AIG1/ADTRP family. The key difference is that TMEM164 utilizes a cysteine-based catalytic mechanism rather than the threonine/serine-based mechanisms found in these other families .

What are the optimal expression systems for producing recombinant bovine TMEM164?

For recombinant production of mammalian transmembrane proteins like TMEM164, mammalian expression systems (particularly HEK293 or CHO cells) often yield the most properly folded and functionally active protein. These systems provide appropriate post-translational modifications and membrane insertion machinery. For preliminary studies, insect cell systems (Sf9 or High Five) may offer a balance between proper folding and higher yields. When designing expression constructs, consider incorporating:

• A cleavable signal peptide for proper membrane targeting

• Affinity tags (6xHis or FLAG) positioned to avoid interference with the catalytic site

• Fluorescent protein fusions for localization studies if relevant

Careful optimization of expression conditions including temperature (typically 30-37°C), induction time, and media composition will be necessary for maximum yield .

What are the challenges in purifying functional TMEM164?

Purification of TMEM164 presents several challenges due to its integral membrane nature. Recommended approaches include:

• Detergent screening: Test multiple detergents (DDM, LMNG, GDN) for extraction efficiency while maintaining enzyme activity

• Two-step purification: Combine immobilized metal affinity chromatography (IMAC) with size exclusion chromatography (SEC)

• Activity preservation: Include appropriate lipids (particularly PC species) during purification to maintain structural integrity

• Consider nanodiscs or liposome reconstitution for downstream functional assays

Validation of proper folding should be performed using circular dichroism and thermal shift assays to ensure the purified protein retains its native conformation .

How can I measure the acyltransferase activity of recombinant TMEM164?

The acyltransferase activity of TMEM164 can be assessed through multiple complementary approaches:

• LC-MS/MS lipidomic analysis: Quantify formation of C20:4-containing ether phospholipids, particularly ePEs. This provides the most comprehensive assessment of specificity and activity.

• Radiometric assay: Using radiolabeled substrates ([14C]-PC or [3H]-lyso-ePE) to measure transfer of labeled acyl chains.

• Fluorescence-based assays: Employ fluorescently labeled lipid substrates to monitor reaction kinetics in real-time.

Activity should be assessed using the physiological substrates C20:4-PC as the acyl donor and various lyso-ePE species as acceptors. Include controls with the C123A mutation (based on human sequence) to confirm specificity of the measured activity .

What is the substrate specificity of TMEM164?

TMEM164 exhibits strong preference for C20:4 (arachidonic acid) transfer, distinguishing it from other acyltransferases. When evaluating substrate specificity, consider:

Acyl chain donors:

• Primary: C20:4-PC (arachidonic acid-containing PC)

• Test alternatives: Other PUFA-PCs (C22:4, C22:6)

• Negative controls: Saturated or monounsaturated PC species

Acyl chain acceptors:

• Primary: Lyso-ether phosphatidylethanolamines (lyso-ePEs)

• Secondary: Lyso-ether phosphatidylcholines (lyso-ePCs)

Reaction parameters:

• pH optimum (likely 7.0-7.5)

• Cation requirements (Ca2+, Mg2+)

• Membrane composition effects

Present results in tabular format showing relative activity across substrate combinations to demonstrate the high selectivity for C20:4 incorporation into ether lipids .

How does TMEM164 contribute to ferroptosis?

TMEM164 plays a crucial role in promoting ferroptosis through multiple mechanisms:

• Generation of oxidation-susceptible lipids: TMEM164 produces C20:4 ether phospholipids (particularly ePEs) that are highly susceptible to peroxidation during ferroptosis.

• Autophagy-dependent mechanisms: TMEM164 specifically supports autophagosome formation during ferroptosis (but not starvation-induced autophagy) by:

  • Facilitating the binding between ATG5 and ATG16L1

  • Supporting the ATG5-dependent autophagosome formation pathway

  • Enabling degradation of critical ferroptosis regulators (GPX4, FTH1)

• Regulation of cellular iron metabolism: TMEM164 influences Fe2+ accumulation, facilitating the Fenton reaction that generates lipid peroxides.

These combined activities position TMEM164 as a master regulator connecting lipid metabolism, autophagy, and ferroptotic cell death pathways .

What methodologies can be used to study TMEM164's role in ferroptosis?

To investigate TMEM164's contribution to ferroptosis, researchers should employ a multi-faceted approach:

Cell viability assays:

• Measure sensitivity to ferroptosis inducers (erastin, RSL3, ML210, sulfasalazine) in control vs. TMEM164-deficient cells

• Include specific ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) as controls

• Compare to non-ferroptotic cell death inducers (staurosporine, TNFα)

Lipid peroxidation measurements:

• BODIPY-C11 staining and flow cytometry for live-cell analysis

• MDA (malondialdehyde) quantification for endpoint assessment

• MS-based quantification of oxidized phospholipids

Iron metabolism:

• Labile iron pool measurements (Calcein-AM quenching)

• Ferrous iron (Fe2+) quantification (FerroOrange staining)

• Iron regulatory protein expression analysis

Autophagy monitoring:

• LC3-II formation by western blot and immunofluorescence

• ATG5-ATG16L1 complex formation by co-immunoprecipitation

• Autophagic flux assessment with lysosomal inhibitors

Genetic manipulation approaches:

• CRISPR-Cas9 knockout of TMEM164

• Rescue experiments with wild-type vs. catalytic mutant (C123A) TMEM164

• Structure-function analysis with domain-specific mutants

These complementary approaches will provide comprehensive insights into TMEM164's mechanistic role in ferroptosis .

How can I design TMEM164 mutants to investigate its structure-function relationship?

Based on structural predictions and functional data, strategic mutations can illuminate TMEM164's mechanism:

Catalytic site mutations:

• C123A (human reference): Predicted to abolish acyltransferase activity

• H181A (human reference): Predicted co-catalytic residue

• Consider equivalent positions in bovine TMEM164 based on sequence alignment

Substrate binding pocket modifications:

• Identify residues lining the internal cavity using structural models

• Create specificity-altering mutations that might shift preference from C20:4 to other acyl chains

• Design mutations at membrane-facing portals to affect substrate access

Transmembrane domain alterations:

• Chimeric constructs swapping TM domains with related family members

• Helix-breaking mutations to assess structural requirements

Interaction surface mapping:

• Alanine scanning of potential protein-protein interaction surfaces

• Focus on regions potentially involved in ATG5-ATG16L1 binding

Each mutant should be assessed for: protein expression and stability, membrane localization, acyltransferase activity, and ability to promote ferroptosis. Present findings as structure-activity relationship tables correlating specific residues with functional outcomes .

What are the most effective methods for analyzing TMEM164-dependent changes in the lipidome?

Comprehensive lipidomic analysis of TMEM164-dependent changes requires sophisticated analytical approaches:

Sample preparation optimization:

• Employ Bligh-Dyer or MTBE extraction methods optimized for phospholipids

• Consider subcellular fractionation to analyze compartment-specific effects

• Include internal standards for each major lipid class

LC-MS/MS methodology:

• Reverse phase chromatography for acyl chain separation

• Hydrophilic interaction chromatography (HILIC) for head group analysis

• Multiple reaction monitoring (MRM) for targeted analysis of ether phospholipids

• High-resolution MS for discovery-based approaches

Data analysis workflow:

• Identify ether phospholipids based on characteristic fragments

• Quantify changes in C20:4 vs. other acyl chain-containing species

• Analyze remodeling patterns in control vs. TMEM164-manipulated samples

• Correlate lipidomic changes with ferroptosis sensitivity

Visualization strategies:

• Heat maps of fold changes across lipid species

• Principal component analysis to identify major sources of variation

• Pathway enrichment analysis using lipid ontology databases

This comprehensive approach will reveal the specific lipid species regulated by TMEM164 and their relationship to ferroptotic sensitivity .

How does TMEM164 expression correlate with cancer progression and prognosis?

TMEM164 expression patterns show context-dependent relationships with cancer outcomes:

Pancreatic Ductal Adenocarcinoma (PDAC):

• TMEM164 mRNA is significantly upregulated in PDAC compared to normal pancreas

• High TMEM164 expression correlates with improved patient survival

• Expression is mainly confined to ductal cells in certain PDAC patients

• Positive correlation with immune cell infiltration, particularly CD8+ T cells, dendritic cells, and cancer-associated fibroblasts

Lung Adenocarcinoma (LUAD):

• TMEM164 is frequently downregulated in LUAD tissues

• Low expression associates with poor prognosis

• Experimental overexpression inhibits proliferation, migration, and invasion

These findings suggest TMEM164 may function as a tumor suppressor in multiple cancer types, potentially through its role in promoting ferroptosis susceptibility. The correlation with immune infiltration further suggests TMEM164 might influence anti-tumor immunity, possibly by affecting the immunogenicity of cancer cell death .

What methodologies should be used to investigate TMEM164's role in tumor immunity?

To explore TMEM164's potential immunomodulatory functions, researchers should employ these approaches:

In vitro co-culture systems:

• TMEM164-manipulated cancer cells with primary immune cells (T cells, dendritic cells)

• Analysis of immunogenic cell death markers (HMGB1, calreticulin, ATP release)

• Evaluation of dendritic cell maturation and T cell activation markers

In vivo tumor models:

• Syngeneic mouse models with TMEM164-knockout or overexpressing cancer cells

• Flow cytometric analysis of tumor-infiltrating immune populations

• Functional assays of tumor-specific T cell responses

• Combined treatment with immunotherapy agents (checkpoint inhibitors)

Mechanistic investigations:

• Analysis of damage-associated molecular patterns (DAMPs) released during TMEM164-dependent ferroptosis

• Evaluation of lipid mediators that might influence immune function

• Investigation of potential direct interactions between TMEM164 and immune signaling pathways

Clinical correlation studies:

• Multiplex immunohistochemistry of human tumor samples for TMEM164 and immune markers

• Correlation with treatment response, particularly to immunotherapies

• Integration with genomic and transcriptomic data

These approaches will help elucidate whether TMEM164-dependent ferroptosis represents a particularly immunogenic form of cell death that could be therapeutically exploited .

How conserved is TMEM164 across species, and what are the implications for using bovine models?

TMEM164 shows significant evolutionary conservation across mammalian species, suggesting fundamental biological importance:

Structural conservation:

• The six transmembrane (6TM) helical core is preserved across species

• The catalytic Cys/His dyad remains invariant in mammals

• Internal cavity architecture for substrate binding appears conserved

Functional conservation:

• Acyltransferase activity is likely maintained across species

• Role in ferroptosis may be evolutionarily conserved

• Substrate specificity for C20:4 incorporation is expected to be consistent

Species-specific considerations:

• Subtle differences in regulatory regions may affect expression patterns

• Minor variations in the substrate binding pocket could influence kinetic parameters

• Species-specific interacting partners might modify functional outcomes

When using bovine TMEM164 as a model, researchers should conduct thorough sequence alignments with human TMEM164 to identify conservation of key residues. The high conservation suggests findings from bovine models will likely translate to human biology, particularly for basic enzymatic mechanisms and structure-function relationships .

How does TMEM164 relate to other transmembrane protein families in structure and function?

TMEM164 belongs to a broader evolutionary family with interesting structural and functional relationships:

Related protein families:

• AIG1/ADTRP family: Human proteins with a similar 6TM core structure but using a Thr/His catalytic dyad rather than Cys/His

• Bacterial YwaF/YpiA proteins: Maintain the 6TM architecture with variable Ser/Thr/Cys nucleophilic residues paired with conserved His

• Loose cluster of hypothetical proteins: Found in bacteria, archaea, and protozoa with divergent active site residues

Functional implications:

• Common evolutionary origin for membrane-embedded acyltransferases

• Diversification of catalytic mechanisms (Cys vs. Thr vs. Ser)

• Adaptation to different lipid environments and metabolic contexts

Research applications:

• Potential to use bacterial homologs for structural studies due to easier expression

• Chimeric constructs between family members to explore determinants of substrate specificity

• Evolutionary analysis to identify conserved functional motifs beyond the catalytic site

Understanding these relationships provides valuable context for interpreting TMEM164 function and may suggest alternative model systems for structural and mechanistic studies .

What are the most promising directions for future TMEM164 research?

Based on current knowledge, these research directions hold particular promise:

• Detailed structural characterization: High-resolution structures through cryo-EM or X-ray crystallography to elucidate the precise catalytic mechanism

• Tissue-specific functions: Investigation of TMEM164's role across different cell types and tissues, particularly in immune cells and the tumor microenvironment

• Development of specific inhibitors/activators: Small molecules targeting TMEM164 to modulate ferroptosis sensitivity in disease contexts

• Intersection with metabolic pathways: Exploration of how TMEM164 activity is regulated by cellular metabolic state and stress conditions

• Therapeutic applications: Leveraging TMEM164's role in ferroptosis for cancer treatment, potentially in combination with immunotherapy

These directions will advance our fundamental understanding of TMEM164 biology while exploring its potential clinical applications .

What technical advancements would facilitate TMEM164 research?

Several technological developments would accelerate progress in TMEM164 research:

• Improved structural tools: Cryo-EM techniques optimized for membrane proteins of TMEM164's size (~30-40 kDa)

• Activity-based probes: Development of chemical probes that specifically label active TMEM164

• Advanced lipidomic workflows: Streamlined protocols for comprehensive analysis of ether phospholipids and their oxidation products

• Ferroptosis biomarkers: Validated markers for monitoring ferroptosis in vivo to track TMEM164-dependent effects

• Animal models: Conditional and tissue-specific TMEM164 knockout/knockin mice to explore physiological functions

• Single-cell technologies: Methods to correlate TMEM164 expression with cellular ferroptosis sensitivity at single-cell resolution