Recombinant Mouse 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma (Agpat3)

What is AGPAT3 and what is its primary function in cellular metabolism?

AGPAT3 (1-acylglycerol-3-phosphate O-acyltransferase 3) is an enzyme that plays a crucial role in glycerophospholipid biosynthesis by converting lysophosphatidic acid (LPA) to phosphatidic acid (PA) . This conversion represents a critical step in the glycerol phosphate pathway for the synthesis of membrane phospholipids. In human studies, AGPAT3 has demonstrated significant acyltransferase activity toward lysophospholipids, with particular specificity for lysophosphatidylinositol (LPI) in the presence of C20:4 fatty acid . The enzyme is involved in lipid metabolism pathways that affect membrane composition and signaling functions through the generation of phosphatidic acid, which serves as both a precursor for phospholipid synthesis and as a lipid second messenger in various signaling cascades .

Where is AGPAT3 primarily localized within cells and what are its expression patterns across tissues?

Studies examining the subcellular localization of AGPAT3 have revealed that when overexpressed, the protein is predominantly detected in the nuclear envelope and the endoplasmic reticulum . This localization pattern is consistent with its role in phospholipid biosynthesis, as these cellular compartments are major sites of membrane lipid production. Unlike AGPAT5, which has been observed to localize in mitochondria when fused with GFP, AGPAT3 does not show significant mitochondrial localization . Regarding tissue distribution, human AGPAT3 mRNA is ubiquitously expressed across tissues, though with notable variations in expression levels . Differential expression patterns between AGPAT3 and its closely related isoform AGPAT4 have been documented, suggesting possible specialized functions in different tissue contexts . This widespread distribution underscores its fundamental importance in cellular lipid metabolism across various tissue types.

How does mouse AGPAT3 differ from human AGPAT3 in terms of structure and function?

While the search results do not provide specific comparative data between mouse and human AGPAT3, general principles of orthologous proteins suggest high conservation in core functional domains while potentially exhibiting species-specific variations in regulatory regions. Based on available information, both human and mouse AGPAT3 function as acyltransferases in the glycerophospholipid biosynthesis pathway . The recombinant mouse AGPAT3 protein (AA 1-376) with His tag is produced using various expression systems, including HEK-293 cells and cell-free protein synthesis methods, suggesting structural similarity that allows for comparable expression strategies . For researchers conducting comparative studies or using mouse models for human disease research, it is advisable to perform sequence alignment analysis to identify conserved functional domains and any species-specific variations that might affect experimental interpretations. When extrapolating findings from mouse models to human applications, these potential differences should be carefully considered.

What are the optimal methods for cloning and expressing recombinant mouse AGPAT3?

For effective cloning and expression of recombinant mouse AGPAT3, researchers have successfully employed several methodological approaches. The gene cloning process typically begins with PCR amplification of AGPAT3 cDNA using gene-specific primers designed for the coding sequence . After PCR product purification, researchers often utilize TA-cloning as an intermediate step before subcloning into expression vectors . For expression vector construction, the AGPAT3 sequence can be excised from the recombinant T-vector using appropriate restriction enzymes (such as APA1 and KPN1) and ligated into mammalian expression vectors like pcDNA3.1+ . For bacterial transformation, competent DH5-Alpha cells and the heat shock method have proven effective, with transformed bacteria selected on LB agar containing ampicillin . Confirmation of successful cloning should include colony PCR and Sanger sequencing to verify the accuracy of the cloned AGPAT3 sequence .

For expression systems, multiple options exist depending on research needs. For protein production, HEK-293 cells have been successfully used to express recombinant mouse AGPAT3 with His tag . Cell-free protein synthesis (CFPS) systems represent an alternative approach that can yield protein with purity levels of approximately 70-80% as determined by SDS-PAGE, Western blot, and analytical SEC methods . For functional studies in mammalian cells, transient transfection using lipid-based transfection reagents such as Lipofectamine 3000 has proven effective, with optimal DNA:reagent ratios depending on the specific application and cell culture format .

What are the recommended approaches for detecting AGPAT3 expression in experimental systems?

For reliable detection and quantification of AGPAT3 expression, researchers should employ a multi-faceted approach combining both nucleic acid and protein-based methods. At the RNA level, reverse transcription quantitative PCR (RT-qPCR) using DNase-treated RNA samples has been successfully applied to confirm AGPAT3 overexpression in transfected cells . This approach is particularly valuable for comparative expression studies and initial validation of genetic manipulations. For designing RT-qPCR primers, targeting conserved regions of the AGPAT3 coding sequence is recommended to ensure specificity and efficiency .

At the protein level, Western blot analysis using antibodies against AGPAT3 or epitope tags (such as V5 or His) for recombinant proteins provides direct evidence of expression . When generating epitope-tagged constructs, positioning the tag at the amino-terminus has been successfully implemented, as demonstrated in the V5-epitope tagging approach for AGPAT3 . For preparing recombinant adenovirus expressing tagged AGPAT3, the AdEasy adenoviral system has proven effective, with viral pools selected based on enzymatic activity for further amplification and purification .

For assessment of subcellular localization, immunofluorescence microscopy or fluorescently tagged fusion proteins can be employed. GFP fusion proteins have been successfully used to visualize the subcellular distribution of AGPAT family members, revealing localization patterns in cellular compartments such as the nuclear envelope, endoplasmic reticulum, and in some cases, mitochondria .

What assays are available for measuring AGPAT3 enzymatic activity, and what are their limitations?

To assess AGPAT3 enzymatic activity, researchers have developed specific acyltransferase assays that measure the conversion of lysophospholipid substrates to their corresponding phospholipid products. These assays typically involve preparing cell lysates from systems overexpressing AGPAT3, such as AD293 cells infected with AGPAT3 recombinant adenovirus . The core enzymatic activity measurement involves quantifying the incorporation of acyl groups from acyl-CoA donors (like arachidonoyl-CoA) into lysophospholipid acceptors (such as lysophosphatidic acid) .

AGPAT3 has demonstrated significant enzymatic activity with an apparent Vmax of approximately 6.35 nmol/min/mg protein for lysophosphatidic acid (LPA) . Importantly, AGPAT3 shows substrate specificity variations, with notable activity toward lysophosphatidylinositol (LPI) when using C20:4 fatty acid . This substrate preference distinguishes it from other AGPAT family members like AGPAT5, which exhibits greater activity toward lysophosphatidylethanolamine (LPE) with C18:1 fatty acid .

Limitations of these assays include:

• Potential interference from endogenous acyltransferases in cell lysates

• Challenges in maintaining optimal enzyme activity during cell lysis and assay procedures

• Variations in substrate availability and solubility affecting reaction kinetics

• Difficulty in distinguishing between closely related AGPAT isoforms with overlapping substrate preferences

To address these limitations, researchers should include appropriate controls, such as lysates from cells expressing empty vectors, and consider using purified recombinant enzymes when possible for more precise kinetic measurements.

How does AGPAT3 participate in the mTORC1 signaling pathway and what are the implications for cisplatin resistance?

Recent research published in March 2025 has revealed a significant role for AGPAT3 in the mTORC1 signaling pathway with important implications for cisplatin resistance in cancer cells . AGPAT3 overexpression in A2780 ovarian cancer cells leads to increased phosphorylation of mTOR, indicating activation of the mTORC1 signaling pathway . Western blot analysis has demonstrated that while total mTOR protein levels may not dramatically increase, the ratio of phosphorylated mTOR to total mTOR significantly increases following AGPAT3 overexpression . Similarly, increased phosphorylation of S6K, a downstream target of mTORC1, has been observed despite decreased total S6K protein levels .

The mechanism connecting AGPAT3 to mTORC1 activation may involve the phospholipase D signaling pathway, as bioinformatic analysis has identified phospholipase D as one of the potential signaling pathways associated with cisplatin chemoresistance . The KEGG database has revealed an interaction between phospholipase D and the mTOR signaling pathway, with AGPAT (also known as LPAAT) playing a role in this network .

The implications for cisplatin resistance are substantial, as mTORC1 hyperactivation is associated with decreased cisplatin-induced apoptosis . This suggests that AGPAT3 could serve as a potential therapeutic target for overcoming cisplatin resistance in cancer treatment. Researchers investigating this pathway should consider the following experimental approaches:

• Combination treatments with mTOR inhibitors and cisplatin in AGPAT3-overexpressing cells

• Assessment of apoptotic markers following AGPAT3 manipulation and cisplatin treatment

• Analysis of phospholipid profiles to elucidate the lipid mediators connecting AGPAT3 activity to mTOR signaling

• Investigation of transcriptional regulation of AGPAT3 in cisplatin-resistant cancer cells

What cell cycle and apoptotic effects are associated with AGPAT3 modulation in experimental systems?

AGPAT3 modulation has been demonstrated to significantly impact cell cycle progression and apoptotic responses in experimental systems. Flow cytometric analysis using propidium iodide (PI) staining has been employed to assess cell cycle distribution (Sub-G1, G1, S, and G2/M phases) in cells with altered AGPAT3 expression . Similarly, apoptosis assays using Annexin-V-FITC/PI staining have revealed altered apoptotic and viable cell rates following AGPAT3 overexpression .

In A2780 ovarian cancer cells, AGPAT3 overexpression has been shown to influence cellular responses to cisplatin treatment, with implications for chemoresistance mechanisms . While the specific details of cell cycle alterations are not fully elaborated in the available search results, the methodological approach using flow cytometry provides a robust framework for investigating these effects.

Researchers studying the impact of AGPAT3 on cell cycle and apoptosis should consider implementing the following experimental design elements:

• Time-course analyses following AGPAT3 modulation to capture dynamic changes

• Comparison of effects under normal growth conditions versus stress conditions (e.g., drug treatment, nutrient deprivation)

• Complementary assessment of cell cycle regulators and apoptotic pathway components using protein and gene expression analyses

• Correlation of cell cycle and apoptotic effects with changes in phospholipid metabolism

The connection between AGPAT3's enzymatic function in phospholipid biosynthesis and its effects on cell cycle and apoptosis suggests complex interplay between lipid metabolism and cellular fate decisions that warrants further investigation.

What is the evidence for AGPAT3 involvement in cancer drug resistance mechanisms beyond cisplatin?

While the search results primarily focus on AGPAT3's role in cisplatin resistance, the underlying mechanisms suggest potential involvement in broader drug resistance phenomena. The identification of AGPAT3 as a differentially expressed gene between cisplatin-sensitive A2780 and cisplatin-resistant A2780cp cell lines, with a substantial log2 fold change of 5.13, indicates its significance in the resistance phenotype . The connection to the mTORC1 pathway is particularly noteworthy, as mTOR signaling is implicated in resistance to various cancer therapeutics beyond platinum compounds.

The phospholipase D signaling pathway, which appears to involve AGPAT3, has been identified through enrichment analysis as associated with cisplatin chemoresistance . This pathway's interaction with mTOR signaling suggests a potential mechanism that could mediate resistance to multiple drugs that trigger apoptotic pathways. Given that AGPAT3 overexpression leads to mTORC1 activation and potentially decreased drug-induced apoptosis, it is reasonable to hypothesize that similar mechanisms might operate in resistance to other apoptosis-inducing agents.

For researchers investigating AGPAT3's potential role in broader drug resistance mechanisms, the following approaches would be valuable:

• Comparative analysis of AGPAT3 expression in cell lines resistant to different classes of chemotherapeutic agents

• Testing whether AGPAT3 overexpression confers cross-resistance to multiple drug classes

• Investigation of whether AGPAT3 inhibition can sensitize cells to various cancer therapeutics

• Exploration of the phospholipid metabolites generated by AGPAT3 activity and their potential roles in cell survival pathways

Currently, direct evidence for AGPAT3's role in non-cisplatin drug resistance is limited in the available search results, highlighting an important area for future research.

What approaches can be used for AGPAT3 gene silencing, and how effective are they?

For effective AGPAT3 gene silencing, researchers have successfully employed short hairpin RNA (shRNA) technology . The design process for AGPAT3-targeting shRNA involves careful analysis of the coding sequence (CDS) structure. In documented approaches, the CDS of AGPAT3 was submitted to the UNAFold web server (unafold.org) for mRNA structural analysis and folding to identify optimal shRNA target positions . Based on such analysis, shRNA constructs can be designed with complementary primers that, when annealed via PCR, form a dimer and complete polymerization to create the final shRNA construct .

For delivery of shRNA constructs, vectors such as pRNA have been successfully used . Transient transfection of these constructs using lipid-based transfection reagents like Lipofectamine 3000 has demonstrated effectiveness in downregulating AGPAT3 expression . When working with cells that express high levels of AGPAT3, such as cisplatin-resistant A2780cis cells, researchers have successfully applied this approach and confirmed downregulation through RT-qPCR and Western blotting analyses .

The effectiveness of gene silencing should be evaluated using multiple complementary methods:

• RT-qPCR to quantify reduction in AGPAT3 mRNA levels

• Western blotting to confirm decreased AGPAT3 protein expression

• Functional assays to verify reduced AGPAT3 enzymatic activity

• Phenotypic assays to assess the biological impact of AGPAT3 knockdown

When designing experiments involving AGPAT3 silencing, researchers should include appropriate controls (scrambled shRNA sequences) and consider potential off-target effects by conducting transcriptome-wide analyses following knockdown.

What are the key considerations for studying AGPAT3 interactions with other proteins and lipid substrates?

Studying AGPAT3 interactions with proteins and lipid substrates requires careful consideration of the enzyme's biochemical properties and cellular localization. AGPAT3 demonstrates substrate specificity that distinguishes it from other AGPAT family members, with significant activity toward lysophosphatidylinositol (LPI) when using C20:4 fatty acid . This substrate preference suggests potential functional specialization in phosphatidylinositol metabolism, which has important implications for signaling pathways.

For investigating protein-protein interactions, researchers should consider:

• Subcellular localization constraints, given AGPAT3's presence in the nuclear envelope and endoplasmic reticulum

• Potential interactions with other enzymes in the glycerophospholipid biosynthesis pathway

• Associations with components of the mTOR signaling complex, given the functional connection to mTORC1 activation

• Possible regulatory interactions that modulate AGPAT3 enzymatic activity

Methodological approaches for studying these interactions may include:

• Co-immunoprecipitation followed by mass spectrometry for unbiased identification of interaction partners

• Proximity labeling techniques (BioID, APEX) to identify neighboring proteins in native cellular environments

• Fluorescence resonance energy transfer (FRET) to detect direct protein-protein interactions in living cells

• Lipidomic analyses to characterize changes in phospholipid profiles following AGPAT3 modulation

For lipid substrate interactions, in vitro enzymatic assays with purified or semi-purified AGPAT3 protein and various lysophospholipid substrates can provide valuable insights into substrate preferences and kinetic parameters . When designing such experiments, researchers should carefully consider the presentation of lipid substrates, as the physical state of these amphipathic molecules can significantly impact enzyme accessibility and activity.

How can researchers develop effective inhibitors or modulators of AGPAT3 activity for experimental applications?

Developing effective inhibitors or modulators of AGPAT3 activity represents an important frontier in research, particularly given its emerging role in cisplatin resistance through mTORC1 pathway modulation . While the search results do not directly address inhibitor development, several approaches can be inferred from the available information on AGPAT3 structure and function.

Structure-based design approaches would benefit from:

• Detailed structural information about AGPAT3's active site and substrate binding pockets

• Molecular modeling of interactions between AGPAT3 and its substrates (LPA, LPI)

• Identification of structural features that distinguish AGPAT3 from other AGPAT family members to enable isoform selectivity

High-throughput screening approaches could include:

• Development of cell-based assays monitoring AGPAT3 activity through changes in phospholipid profiles

• In vitro enzymatic assays using purified or semi-purified AGPAT3 protein and fluorescent or radioactive substrates

• Phenotypic screens in cancer cell lines where AGPAT3 modulation affects drug sensitivity

Genetic approaches for tool development might involve:

• CRISPR-Cas9-mediated generation of AGPAT3 variants with altered activity for structure-function studies

• Creation of dominant-negative AGPAT3 mutants for acute inhibition in experimental systems

• Inducible expression systems for temporal control of AGPAT3 expression

Given AGPAT3's role in the mTORC1 pathway and cisplatin resistance, combination approaches targeting both AGPAT3 and mTOR signaling components might provide synergistic effects in experimental settings . Any developed inhibitors should be carefully characterized for specificity against other AGPAT family members and related acyltransferases to ensure target selectivity.

What analytical methods are recommended for interpreting AGPAT3 expression data in different experimental contexts?

For robust interpretation of AGPAT3 expression data across experimental contexts, researchers should employ multiple complementary analytical approaches. Statistical analysis using appropriate software such as GraphPad Prism has been successfully applied to analyze AGPAT3 expression data, with Student's t-test employed for comparing two normally distributed variables . When analyzing differential expression between experimental conditions (such as drug-sensitive versus resistant cell lines), volcano plots and principal component analysis (PCA) can provide valuable visualization of the data landscape .

For RNA-sequencing data analysis, careful attention to batch effect removal is essential to eliminate biases introduced by experimental batches, as demonstrated in the analysis of AGPAT3 expression in cisplatin-resistant cell lines . When analyzing AGPAT3 expression in relation to other genes, heatmap visualization can effectively represent relative expression patterns across multiple genes and conditions .

Key considerations for data interpretation include:

• Establishing appropriate thresholds for significance (e.g., p-adj < 0.05, |log2(FC)| > 1) when identifying differentially expressed genes

• Using base mean expression values to filter for genes with sufficient expression levels (e.g., base mean > 50) to increase reliability

• Performing pathway enrichment analysis to contextualize AGPAT3 expression changes within broader biological processes

• Correlating mRNA expression data with protein levels through complementary Western blot analysis

When comparing AGPAT3 expression across tissue types or experimental conditions, normalization to appropriate housekeeping genes or internal controls is critical for accurate interpretation . For protein-level analysis, quantification relative to loading controls such as β-actin enables meaningful comparisons .

How can researchers effectively integrate AGPAT3 findings into broader phospholipid metabolism studies?

Integrating AGPAT3 findings into broader phospholipid metabolism studies requires consideration of its position within lipid biosynthetic pathways and its unique substrate preferences. AGPAT3's significant acyltransferase activity toward lysophosphatidylinositol (LPI) in the presence of C20:4 fatty acid distinguishes it from other AGPAT family members and suggests specialized roles in phosphatidylinositol metabolism . This substrate preference has important implications for signaling pathways, as phosphoinositides are critical regulators of numerous cellular processes.

For effective integration, researchers should consider:

• Conducting comprehensive lipidomic analyses to map changes in multiple phospholipid species following AGPAT3 modulation

• Correlating AGPAT3 activity with levels of bioactive lipid mediators derived from its products

• Investigating crosstalk between AGPAT3-mediated phospholipid synthesis and other lipid metabolic pathways

• Examining the impact of AGPAT3-generated phospholipids on membrane properties and organization

The connection between AGPAT3 and the mTORC1 signaling pathway provides an important link between phospholipid metabolism and cellular signaling networks . This relationship can be explored through integrated analyses of:

• Phospholipid changes following mTORC1 modulation

• Effects of specific phospholipid species on mTORC1 activity

• Lipid composition of cellular compartments where mTORC1 signaling occurs

• Temporal relationships between AGPAT3 activity, phospholipid changes, and mTORC1 activation

The subcellular localization of AGPAT3 in the nuclear envelope and endoplasmic reticulum further informs its integration into phospholipid metabolism studies, as these compartments have distinct lipid composition requirements and biosynthetic capacities .

What are the most promising future research directions for AGPAT3 in cancer and drug resistance studies?

Based on recent findings, several promising research directions emerge for investigating AGPAT3's role in cancer and drug resistance:

• Mechanistic Studies of mTORC1 Activation: Further elucidation of the molecular mechanisms connecting AGPAT3 activity to mTORC1 activation would provide valuable insights into cancer cell signaling and potential therapeutic targets . This could include identification of specific phospholipid species that mediate this connection and characterization of the intermediate signaling components.

• Biomarker Development: Given the significant upregulation of AGPAT3 in cisplatin-resistant cells (log2(FC) of 5.13), investigation of AGPAT3 as a potential biomarker for predicting chemotherapy response in clinical samples represents an important translational direction . This could involve retrospective analysis of patient samples and correlation with treatment outcomes.

• Combination Therapy Approaches: Testing combinations of AGPAT3 inhibition with conventional chemotherapeutics or mTOR pathway inhibitors could yield synergistic effects in overcoming drug resistance . This direction would benefit from the development of specific AGPAT3 inhibitors or genetic approaches for AGPAT3 targeting.

• Broader Cancer Type Exploration: Extending the investigation of AGPAT3's role in drug resistance beyond ovarian cancer to other malignancies would establish the generalizability of these findings. This could include analysis of AGPAT3 expression across cancer types and correlation with treatment response data.

• Lipid Metabolism Reprogramming: Comprehensive characterization of how AGPAT3 overexpression reprograms cancer cell lipid metabolism could reveal additional vulnerabilities for therapeutic targeting. This would involve detailed lipidomic analyses and functional studies of lipid-dependent processes.

• Translational Models: Development of in vivo models with modulated AGPAT3 expression would enable testing of its role in tumor growth, metastasis, and treatment response in more physiologically relevant contexts. This could include patient-derived xenografts with varying AGPAT3 expression levels.

These research directions hold potential for advancing our understanding of cancer biology and developing novel therapeutic strategies to overcome drug resistance, with AGPAT3 emerging as a promising target at the intersection of lipid metabolism and cancer cell signaling.