CES1 Antibody

What is CES1 and what biological functions does it perform?

CES1 (Carboxylesterase 1) is a member of the serine hydrolase superfamily that catalyzes the hydrolysis of ester and amide bonds across a broad spectrum of substrates. It plays several critical biological roles:

• Drug metabolism: CES1 contributes to approximately 90% of hepatic hydrolytic activity in human livers, making it a major determinant of pharmacokinetic behavior for therapeutic agents containing ester groups .

• Detoxification: It participates in detoxification of drugs such as cocaine and heroin in serum and liver, with the resulting de-esterified metabolites being secreted in urine .

• Chemical defense: CES1 can detoxify organophosphate and carbamate analogues found in agrochemicals or chemical nerve agents (e.g., malathion, sarin, tabun, and VX) .

• Lipid metabolism: Beyond hydrolytic activity, CES1 performs transesterification reactions important for cholesterol homeostasis .

• Immune regulation: Recent research demonstrates CES1's role in shaping dendritic cell metabolic profiles and function, influencing their inflammatory phenotype and T cell polarization capacity .

CES1 is a 62 kDa protein with 567 amino acids and is primarily expressed in liver tissue and mononuclear myeloid cells, with particularly high expression in monocytes and macrophages .

How should I select an appropriate CES1 antibody for my research application?

Selecting the appropriate CES1 antibody depends on several key factors:

1. Experimental application:

• For Western blot: Both monoclonal and polyclonal antibodies work well, with typical dilutions ranging from 1:500-1:6000 .

• For immunohistochemistry: Polyclonal antibodies often provide better sensitivity with dilutions of 1:50-1:500 .

• For immunofluorescence: Consider antibodies validated specifically for IF with recommended dilutions of 1:200-1:800 .

2. Species reactivity requirements:

• Human-specific: For human-only studies, antibodies like MAB4920 (clone #544623) are optimized for human CES1 .

• Multi-species: For comparative studies, antibodies with broader reactivity like AF4920 (reacts with human, mouse, and rat) are preferable .

3. Target region and specificity:

• Some antibodies target specific regions, such as His19-Glu563 in human CES1 .

• Consider antibodies raised against recombinant fusion proteins containing sequences corresponding to specific amino acid regions of CES1 .

4. Validation data:

• Review Western blot images showing expected molecular weight (approximately 60-70 kDa for CES1) .

• Check immunohistochemistry or immunofluorescence validation in relevant tissues (particularly liver) .

5. Host species considerations:

• Available host species include rabbit, mouse, sheep, and goat .

• Choose a host species that minimizes cross-reactivity with other antibodies in your experimental system.

Always validate the antibody in your specific experimental conditions, as optimal dilutions may vary depending on sample type and preparation methods .

What detection methods can I use with CES1 antibodies and what are their limitations?

CES1 antibodies can be utilized across multiple detection methods, each with specific advantages and limitations:

1. Western Blot (WB):

• Protocol highlights: Use PVDF membrane with 1-2 μg/mL antibody concentration for human samples . For mouse samples, 1 μg/mL is typically sufficient .

• Expected results: CES1 appears as a specific band at approximately 60-70 kDa (human: ~62-70 kDa, mouse: ~60 kDa) .

• Limitations: May require reducing conditions and specific buffer groups (e.g., Immunoblot Buffer Group 1) .

• Sample preparation: Works well with lysates from liver tissue, HepG2, THP-1, and Caco-2 cells .

2. Simple Western™ (automated capillary Western):

• Protocol highlights: Load liver tissue at 0.2 mg/mL with 2-10 μg/mL antibody concentration .

• Expected results: A specific band at approximately 62-65 kDa .

• Advantages: More precise quantification with lower sample requirements.

• Limitations: Requires specialized equipment and optimization.

3. Immunohistochemistry (IHC):

• Protocol highlights: Typical dilutions of 1:50-1:500 with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

• Optimal tissues: Validated in mouse liver tissue .

• Limitations: May show background staining; requires careful optimization of antigen retrieval methods.

4. Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Protocol highlights: Typical dilutions of 1:200-1:800 .

• Validated cell lines: C2C12 and HepG2 cells .

• Limitations: Autofluorescence in some tissues can interfere with signal detection.

5. ELISA:

• Applications: Available with conjugated antibodies (HRP, FITC, or Biotin) .

• Limitations: May require specific conjugated antibodies for optimal sensitivity.

Detection troubleshooting:

• For weak signals: Increase antibody concentration, extend incubation time, or use more sensitive detection systems.

• For high background: Increase blocking time/concentration, optimize washing steps, or use more specific secondary antibodies.

• For multiple bands: Verify sample preparation conditions, use freshly prepared samples, or consider alternative antibody clones.

Each detection method requires specific optimization for your experimental system to achieve optimal signal-to-noise ratio .

What is the optimal storage and handling protocol for CES1 antibodies?

Proper storage and handling of CES1 antibodies are critical for maintaining their activity and specificity over time:

Storage conditions:

• Long-term storage: Store at -20°C to -70°C for up to 12 months from the date of receipt .

• Medium-term storage: For reconstituted antibodies, store at 2-8°C under sterile conditions for up to 1 month .

• Extended storage after reconstitution: Can be stored at -20°C to -70°C under sterile conditions for up to 6 months .

Handling guidelines:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain antibody integrity .

• For small volume antibodies (e.g., 20μL), aliquoting is unnecessary for -20°C storage, but larger volumes should be aliquoted to minimize freeze-thaw cycles .

• Some formulations contain preservatives like 0.02% sodium azide and stabilizers like 50% glycerol (pH 7.3) or 0.1% BSA .

Reconstitution protocol:

• Briefly centrifuge the antibody vial before opening.

• Reconstitute lyophilized antibodies according to manufacturer's specifications.

• Mix gently to avoid foaming and microbial contamination.

• Allow the reconstituted antibody to sit at room temperature for 30 minutes before use or storage.

Working solution preparation:

• Dilute antibodies in appropriate buffers immediately before use.

• For Western blot applications with human liver samples, typical concentrations range from 2-10 μg/mL .

• For immunohistochemistry applications, dilutions typically range from 1:50-1:500 .

Quality control considerations:

• Monitor antibody performance through consistent positive controls.

• Include negative controls to assess background and non-specific binding.

• Document lot-to-lot variations that may affect experimental outcomes.

These storage and handling protocols help ensure consistent antibody performance across experiments and maximize the shelf-life of your CES1 antibodies .

In which tissues and cell types is CES1 predominantly expressed?

CES1 shows distinct expression patterns across tissues and cell types, which is important knowledge for designing relevant experimental models:

Primary tissue expression:

• Liver: CES1 is abundantly expressed in human, mouse, and rat liver tissue, contributing to approximately 90% of hepatic hydrolytic activity in humans .

• Immune tissues: Significant expression in mononuclear myeloid cells, particularly in monocytes and macrophages .

Cell line expression:

• Hepatic cell lines: Strongly expressed in HepG2 human hepatocellular carcinoma cells .

• Immune cell lines: High expression in THP-1 monocytic cells .

• Intestinal cell lines: Detectable in Caco-2 intestinal epithelial cells .

• Muscle cell lines: Detectable in C2C12 mouse myoblast cells .

Developmental and differentiation patterns:

• CES1 expression peaks early during monocyte to dendritic cell (MoDC) differentiation, suggesting a role in early differentiation processes .

• Expression levels can change in response to inflammatory stimuli and metabolic alterations .

Species-specific expression patterns:

• While CES1 is conserved across mammals, expression levels and tissue distribution can vary between species.

• Human, mouse, and rat all show high hepatic expression, making these useful comparative models .

Experimental implications:

• For studying drug metabolism: Liver tissue or hepatic cell lines (HepG2) are optimal models.

• For immune function studies: THP-1 cells or primary monocytes/macrophages are appropriate.

• For metabolic studies: Consider using liver tissue or cultured hepatocytes.

• For dendritic cell research: Monitor expression changes during differentiation from monocytes .

Understanding these expression patterns helps researchers select appropriate experimental systems and positive controls when studying CES1 function or using CES1 antibodies for detection .

How does CES1 inhibition affect dendritic cell differentiation and inflammatory responses?

CES1 inhibition significantly impacts dendritic cell (DC) differentiation and function, revealing a critical role in immunometabolism and inflammatory responses:

Experimental approach:
Recent research utilized the CES1 inhibitor WWL113 during monocyte-to-DC differentiation (MoDCs) alongside genetic overexpression models to comprehensively examine CES1's role in DC function .

Key phenotypic changes in CES1-inhibited DCs:

• Surface marker expression: Increased expression of CD209, CD86, and MHCII, indicating enhanced maturation and activation potential .

• Cytokine production: Higher secretion of pro-inflammatory cytokines (IL-6, IL-8, TNF) and also IL-10, suggesting a complex immunomodulatory profile .

• Functional capacity: Enhanced phagocytic ability and increased capacity to polarize T helper 17 (Th17) differentiation in autologous DC-T cell coculture models .

Metabolic reprogramming effects:

• Mitochondrial function: Impaired maximal mitochondrial respiration capacity in CES1-inhibited DCs .

• Glycolytic shift: Increased lactate production, indicating a metabolic shift toward glycolysis .

• Metabolite alterations: Decreased intracellular amino acids and tricarboxylic acid cycle intermediates .

Transcriptomic changes:
Comprehensive RNA sequencing revealed enrichment of multiple inflammatory and metabolic pathways in CES1-inhibited DCs, providing mechanistic insights into the observed phenotypic changes .

In vivo relevance:
Transgenic human CES1 overexpression in murine DCs generated:

• A less inflammatory phenotype

• Increased resistance to T cell-mediated colitis
  These findings suggest CES1's potential therapeutic relevance in inflammatory conditions .

Methodological implications for research:

• When studying DC function, researchers should consider CES1 expression levels as a potentially confounding variable.

• Monitoring CES1 expression during DC differentiation experiments is advisable for data interpretation.

• CES1 inhibition could be employed as an experimental approach to generate DCs with enhanced inflammatory and Th17-polarizing properties .

This research establishes CES1 as a metabolic regulator that directs DC differentiation toward specific functional phenotypes, with CES1 inhibition promoting a more inflammatory profile that supports Th17 responses .

What is the role of CES1 in oxylipin release from oxidized triacylglycerol and what are the implications for inflammation research?

CES1 plays a crucial role in releasing bioactive oxylipins from oxidized triacylglycerols (oxTAGs), representing an important intersection between lipid metabolism and inflammatory signaling:

Biochemical mechanism:
Human CES1, as a member of the serine hydrolase superfamily, catalyzes the hydrolysis of oxidized fatty acids from the glycerol backbone of oxTAGs. Specifically, when triacylglycerols containing oxidized polyunsaturated fatty acids (at approximately 5 mol%) are exposed to recombinant CES1, the enzyme releases both oxylipins and non-oxidized fatty acids (arachidonic acid or linoleic acid) .

Oxylipin release profile:
CES1 hydrolysis of oxTAGs yields specific regioisomers of bioactive lipid mediators:

• From arachidonic acid-containing oxTAGs: 5-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs)

• From linoleic acid-containing oxTAGs: 9- and 13-hydroxyoctadecadienoic acids (HODEs)

Cellular consequences of CES1 deficiency:
THP-1 macrophages with reduced CES1 levels show:

• Enhanced accumulation of oxTAGs and TAGs, suggesting impaired lipid metabolism

• Differential responses to extracellular stimuli including:

  • Altered response to oxTAGs

  • Modified reactions to lipopolysaccharide

  • Changed response to 15-HETE exposure

• Distinct cytokine profiles and prostaglandin E2 production

Methodological considerations for researchers:

• When studying inflammation in macrophages or related cell types, CES1 expression levels should be monitored as they may influence lipid mediator production.

• Mass spectrometry approaches can be used to profile specific oxylipin species released by CES1 activity.

• CES1 inhibitors could potentially be used to modulate oxylipin release in experimental systems.

Research implications:
This CES1-mediated oxylipin release represents an important mechanism by which oxidized lipids can influence inflammatory responses. The finding positions CES1 as a key enzyme at the interface between lipid metabolism and inflammation, suggesting its potential importance in conditions characterized by both metabolic dysregulation and inflammatory processes .

Understanding this mechanism provides researchers with new perspectives on how lipid oxidation products may influence inflammatory signaling through CES1-dependent processes, opening avenues for therapeutic interventions targeting this pathway .

What are the optimal Western blot conditions for detecting CES1 across different species and sample types?

Achieving consistent and specific detection of CES1 via Western blot requires careful optimization based on species, sample type, and experimental goals:

Sample preparation considerations:

• Human samples: Liver tissue and cell lines (HepG2, THP-1, Caco-2) have shown reliable CES1 detection .

• Mouse samples: Liver tissue provides robust signals, but expression in other tissues may require increased protein loading .

• Sample loading: For liver tissue lysates, 0.2 mg/mL concentration is typically sufficient for detection .

• Lysis buffer selection: Standard RIPA buffer with protease inhibitors is generally effective for CES1 extraction.

Electrophoresis and transfer conditions:

• Reducing conditions: All validated protocols for CES1 detection use reducing conditions .

• Membrane selection: PVDF membrane has been successfully used across multiple studies .

• Transfer parameters: Standard wet transfer protocols at 100V for 1 hour or overnight at 30V at 4°C have proven effective.

Antibody selection and concentration by species:

• Human CES1:

  • Monoclonal antibody (MAB4920, clone #544623): 2 μg/mL

  • Polyclonal antibody (CAB1853): 1:500-1:5000 dilution

• Mouse CES1:

  • Sheep polyclonal antibody (AF7929): 1 μg/mL

  • Cross-reactive polyclonal antibodies: 0.2-2 μg/mL

• Multi-species detection:

  • Goat anti-human/mouse/rat polyclonal antibody (AF4920): 0.2 μg/mL

Detection systems:

• Primary detection: HRP-conjugated secondary antibodies have been validated for all listed primaries .

• Alternative detection: For enhanced sensitivity, consider fluorophore-conjugated secondaries or advanced chemiluminescence substrates.

• Specific secondary antibodies:

  • For mouse primaries: Anti-mouse IgG (HAF007)

  • For sheep primaries: Anti-sheep IgG (HAF016)

  • For goat primaries: Anti-goat IgG (HAF019 or HAF109)

Expected results by species:

• Human CES1: Specific band at approximately 62-70 kDa

• Mouse CES1: Specific band at approximately 60 kDa

• Rat CES1: Specific band at approximately 63 kDa

Buffer systems:

• Immunoblot Buffer Group 1 has been validated for CES1 detection in multiple studies .

Troubleshooting considerations:

• For weak signals: Increase antibody concentration, extend incubation time, or use fresh samples.

• For multiple bands: Verify sample integrity, consider alternative antibody clones, or perform additional blocking steps.

• For high background: Increase BSA concentration in blocking buffer, optimize washing steps, or reduce secondary antibody concentration.

Following these optimized conditions can help ensure specific and consistent detection of CES1 across different experimental systems .

What are the optimal antibody dilutions and conditions for different applications when studying CES1?

Successful CES1 detection across various applications requires specific antibody dilutions and experimental conditions:

1. Western Blot (WB) Applications:

Expected results: Single band at approximately 60-70 kDa depending on species (human: ~62-70 kDa; mouse: ~60 kDa) .

2. Immunohistochemistry (IHC) Applications:

Validated tissues: Liver tissue shows consistent staining across species .

3. Immunofluorescence (IF)/Immunocytochemistry (ICC) Applications:

Counterstaining: DAPI for nuclear visualization .

4. ELISA Applications:

Conjugated antibody options: Available with HRP, FITC, or Biotin conjugates for different detection requirements .

5. Simple Western™ (Automated Capillary Western) Applications:

Expected results: Band at approximately 62-65 kDa .

6. General Optimization Guidelines:

• Initial testing: Start with the manufacturer's recommended dilution for each application.

• Optimization approach: Perform a titration experiment with 3-5 different dilutions.

• Signal-to-noise ratio: Select the dilution that provides the strongest specific signal with minimal background.

• Incubation time adjustment: For weaker signals, consider extending primary antibody incubation (overnight at 4°C).

• Controls: Always include positive controls (liver tissue/cells) and negative controls (antibody omission, isotype controls).

These application-specific parameters have been validated in published studies and manufacturer recommendations, but optimal conditions may require fine-tuning based on your specific experimental system and detection equipment .

How can researchers validate CES1 knockdown or knockout efficiency in experimental models?

Effective validation of CES1 knockdown or knockout is critical for ensuring experimental rigor when studying CES1 function. Here are comprehensive approaches for validation across different model systems:

1. Protein-level validation methods:

• Western blot analysis:

  • Primary antibody selection: Use validated antibodies like MAB4920 (human-specific) or AF4920 (multi-species) .

  • Loading controls: Include appropriate housekeeping proteins (β-actin, GAPDH, α-tubulin).

  • Quantification: Perform densitometric analysis to calculate knockdown efficiency (percentage reduction compared to control).

  • Expected results: Significant reduction in the ~62-70 kDa CES1 band in knockdown/knockout samples .

• Simple Western™ (automated capillary-based Western):

  • Offers increased sensitivity and quantitative precision.

  • Use 0.2 mg/mL sample concentration with 2-10 μg/mL antibody concentration .

  • Provides more accurate quantification of knockdown efficiency.

• Immunocytochemistry/Immunofluorescence:

  • Useful for visualizing cellular distribution and confirming uniform knockdown across cell populations.

  • Use 1:200-1:800 dilution of validated antibodies .

  • Include quantitative image analysis to measure signal intensity reduction.

2. mRNA-level validation methods:

• Quantitative PCR (qPCR):

  • Design primers specific to CES1 (avoiding homologous carboxylesterases).

  • Normalize to stable reference genes (validated in your experimental system).

  • Calculate fold change using the 2^-ΔΔCt method.

  • This approach was successfully used to analyze CES1 expression during monocyte differentiation to DCs .

• RNA sequencing:

  • Provides comprehensive transcriptome analysis to confirm CES1 reduction.

  • Also reveals compensatory gene expression changes that may affect interpretation.

  • Was successfully applied in dendritic cell CES1 inhibition studies .

3. Functional validation approaches:

• Enzymatic activity assays:

  • Substrate hydrolysis: Measure CES1-specific substrate (e.g., para-nitrophenyl acetate) hydrolysis rates.

  • Oxylipin release: Quantify specific oxylipin release from oxTAGs using mass spectrometry .

  • Drug metabolism: Assess conversion rates of CES1-metabolized drugs (e.g., methylphenidate).

• Metabolic phenotyping:

  • Cellular respiration: Measure maximal mitochondrial respiration capacity using Seahorse respirometry .

  • Lactate production: Quantify lactate as a marker of glycolytic shift .

  • Metabolomic analysis: Assess TCA cycle intermediates and amino acid profiles .

4. Phenotypic validation:

• For immune cells:

  • Surface marker expression: Measure CD209, CD86, and MHCII expression on dendritic cells .

  • Cytokine production: Quantify IL-6, IL-8, TNF, and IL-10 secretion .

  • Functional assays: Assess phagocytic capacity and T cell polarization ability .

• For metabolic models:

  • Lipid accumulation: Measure oxTAG/TAG levels in macrophages .

  • Inflammatory response: Assess responses to stimuli like oxTAGs, LPS, or 15-HETE .

These multi-level validation approaches ensure comprehensive confirmation of CES1 knockdown/knockout, critical for attributing observed phenotypes to CES1 deficiency with confidence .

What role does CES1 play in drug metabolism and what implications does this have for pharmacokinetic studies?

CES1 serves as a critical enzyme in drug metabolism with significant implications for pharmacokinetic studies, drug development, and precision medicine approaches:

Mechanism of CES1-mediated drug metabolism:

• CES1 catalyzes the hydrolysis of ester and amide bonds in various drug compounds.

• It contributes to approximately 90% of hepatic hydrolytic activity in human livers, making it a major determinant of drug pharmacokinetics .

• Beyond simple hydrolysis, CES1 can perform transesterification reactions, which further impacts drug metabolism processes .

Classes of medications metabolized by CES1:

• Prodrugs requiring activation:

  • Certain medications are administered as inactive esters that require CES1-mediated hydrolysis to release the active compound.

  • Examples include some angiotensin-converting enzyme inhibitors and antiviral prodrugs.

• Drugs inactivated by CES1:

  • Many therapeutic agents are inactivated through CES1-mediated de-esterification.

  • This mechanism contributes to drug clearance and termination of therapeutic effects.

• Drugs of abuse:

  • CES1 participates in the detoxification of drugs such as cocaine and heroin in serum and liver.

  • The resulting de-esterified metabolites are more water-soluble and can be excreted in urine .

• Chemical and environmental compounds:

  • CES1 can detoxify organophosphate and carbamate compounds found in agrochemicals.

  • It also metabolizes chemical nerve agents like malathion, sarin, tabun, and VX .

Implications for pharmacokinetic studies:

• Inter-individual variability:

  • Variation in CES1 expression or activity can significantly affect drug metabolism rates.

  • Studies have demonstrated that CES1 variation can affect the metabolism of medications like methylphenidate .

• Species differences:

  • While CES1 is conserved across mammals, there are species-specific differences in activity and substrate specificity.

  • This necessitates careful interpretation when translating findings from animal models to humans.

  • Researchers should consider that mouse CES1 (~60 kDa) and human CES1 (~62-70 kDa) may have different substrate profiles .

• Tissue-specific metabolism:

  • CES1 is predominantly expressed in liver, but also found in macrophages and other cell types.

  • This distribution affects the sites of drug metabolism and local drug concentrations .

• Drug-drug interactions:

  • Medications competing for CES1-mediated metabolism may interact, affecting plasma concentrations.

  • CES1 inhibitors can potentially alter the pharmacokinetics of numerous medications.

Methodological considerations for researchers:

• When studying drug metabolism, monitoring CES1 expression and activity is essential.

• In cell models, CES1 levels should be verified using validated antibodies (e.g., via Western blot at 1:1000-1:6000 dilution) .

• For in vivo studies, species differences in CES1 activity should be considered when translating findings.

• CES1 genetic polymorphisms may need to be assessed in clinical pharmacokinetic studies to explain variability.

Understanding CES1's role in drug metabolism provides critical insights for drug development, pharmacokinetic modeling, and personalized medicine approaches .

How does post-translational modification affect CES1 function and antibody recognition?

Post-translational modifications (PTMs) of CES1 significantly impact both its enzymatic function and antibody recognition, creating important considerations for experimental design and data interpretation:

Common PTMs affecting CES1:

• Glycosylation:

  • CES1 contains several potential N-glycosylation sites.

  • Glycosylation can affect protein folding, stability, and enzymatic activity.

  • Impact on antibody recognition: Heavily glycosylated forms may show altered molecular weight (appearing at 62-70 kDa rather than the predicted 62 kDa) .

• Phosphorylation:

  • Serine, threonine, and tyrosine residues in CES1 can undergo phosphorylation.

  • This modification can modulate enzyme activity and protein-protein interactions.

  • Impact on antibody recognition: Phosphorylation may mask epitopes recognized by some antibodies.

• Ubiquitination:

  • Regulates CES1 protein turnover and degradation.

  • Impact on antibody recognition: Ubiquitinated forms may appear as higher molecular weight bands in Western blots.

Effects on experimental detection:

• Western blot considerations:

  • Observed molecular weight variations: CES1 is detected between 60-70 kDa across different studies .

  • Human liver samples show CES1 at approximately 70 kDa in some studies and 63 kDa in others .

  • Mouse liver samples typically show CES1 at approximately 60 kDa .

  • These variations may reflect different PTM patterns or detection conditions.

• Antibody selection strategies:

  • Epitope location: Consider whether the antibody targets regions susceptible to PTMs.

  • For studies focused on specific PTM forms, select antibodies validated to recognize those forms.

  • Multi-antibody approach: Using antibodies targeting different epitopes can provide comprehensive detection.

• Sample preparation effects:

  • Denaturing conditions: Reducing conditions are consistently recommended for CES1 detection .

  • Buffer systems: Immunoblot Buffer Group 1 has been validated for detecting various CES1 forms .

  • Tissue/cell source: Different tissues may exhibit distinct PTM patterns.

Functional implications of PTMs:

• Enzymatic activity:

  • PTMs can alter substrate specificity and catalytic efficiency.

  • When measuring CES1 activity, consider that protein levels may not directly correlate with activity if PTMs affect function.

• Cellular localization:

  • PTMs may influence CES1's subcellular distribution.

  • This can impact its accessibility to substrates and interaction partners.

• Protein stability:

  • Some PTMs can alter CES1's half-life and degradation rate.

  • This affects steady-state protein levels independent of gene expression.

Methodological recommendations:

• For comprehensive detection:

  • Use antibodies validated to recognize diverse CES1 forms (e.g., AF4920 for multi-species detection) .

  • Consider using multiple antibodies targeting different epitopes.

• For PTM-specific studies:

  • Complement antibody detection with mass spectrometry analysis.

  • Use phosphatase or glycosidase treatments to confirm PTM-dependent detection.

• For functional correlation:

  • Combine protein detection with activity assays to assess PTM impact on function.

  • Consider using recombinant CES1 with defined PTM status as controls.

Understanding the impact of PTMs on CES1 detection and function is essential for accurate experimental design and interpretation in CES1 research .

What are the current methods for measuring CES1 enzymatic activity in biological samples?

Assessing CES1 enzymatic activity in biological samples requires specialized approaches that can distinguish CES1 from other carboxylesterases. Here are the contemporary methodological strategies:

1. Substrate-based colorimetric/fluorometric assays:

• para-Nitrophenyl acetate (p-NPA) hydrolysis:

  • Principle: CES1 hydrolyzes p-NPA to release p-nitrophenol, which can be measured spectrophotometrically at 405 nm.

  • Specificity enhancement: Use CES1-selective inhibitors (e.g., WWL113) as controls to distinguish CES1 activity from other esterases .

  • Applications: Suitable for cell lysates, tissue homogenates, and recombinant enzyme.

  • Limitations: Moderate specificity, as other esterases can also hydrolyze this substrate.

• Fluorescent substrate hydrolysis:

  • Principle: CES1 cleaves ester bonds in fluorogenic substrates, releasing fluorescent products.

  • Examples: 4-Methylumbelliferyl acetate (4-MUA) or specific BODIPY-labeled esters.

  • Advantages: Higher sensitivity than colorimetric methods; some substrates offer improved selectivity.

  • Applications: Cell-based assays, microplate formats, and high-throughput screening.

2. Drug substrate-specific methods:

• HPLC-based drug metabolism assays:

  • Principle: Measure conversion of CES1-specific drug substrates to their metabolites.

  • Example substrates: Methylphenidate, cocaine, or specific prodrugs.

  • Detection: HPLC with UV, fluorescence, or mass spectrometry detection.

  • Advantages: High specificity for CES1 activity with pharmacologically relevant substrates.

  • Applications: Pharmacokinetic studies, enzyme kinetics, inhibitor screening.

3. Lipid metabolism-focused approaches:

• Oxylipin release quantification:

  • Principle: Measure CES1-mediated release of oxylipins from oxidized triacylglycerols (oxTAGs).

  • Methodology: Incubate oxTAGs with samples containing CES1, then quantify released HETEs and HODEs using LC-MS/MS .

  • Applications: Investigating CES1's role in lipid metabolism and inflammatory signaling.

  • Advantages: Physiologically relevant measurement of CES1's activity toward complex lipid substrates .

• Transesterification activity measurement:

  • Principle: Quantify CES1-mediated transfer of acyl groups between substrates.

  • Relevance: Important for cholesterol homeostasis .

  • Detection: LC-MS/MS to monitor formation of transesterification products.

  • Applications: Studies of CES1's role in lipid metabolism beyond simple hydrolysis.

4. Activity-based protein profiling (ABPP):

• Mechanism-based activity probes:

  • Principle: Fluorophosphonate or carbamate probes covalently label the active site of catalytically active CES1.

  • Detection: Gel-based visualization or mass spectrometry-based quantification of labeled enzyme.

  • Advantages: Directly measures catalytically competent enzyme rather than protein abundance.

  • Applications: Inhibitor screening, activity profiling across tissues/conditions.

5. Cell-based functional assays:

• Metabolic phenotyping:

  • Principle: Measure downstream effects of CES1 activity on cellular metabolism.

  • Parameters: Mitochondrial respiration, lactate production, TCA cycle intermediates .

  • Applications: Investigating CES1's role in cellular metabolic programming.

  • Relevance: Successfully applied in dendritic cell models .

Comparative analysis of detection methods:

When selecting a method for measuring CES1 activity, researchers should consider the specific research question, available equipment, required throughput, and need for direct vs. indirect activity measurement .

How does CES1 expression in dendritic cells influence immune function and what are the implications for immunometabolism research?

CES1 expression in dendritic cells (DCs) serves as a critical metabolic checkpoint that shapes immune function, with significant implications for immunometabolism research:

Temporal expression pattern during differentiation:
CES1 expression peaks early during monocyte-to-dendritic cell differentiation, suggesting a regulatory role in the early phases of DC development . This temporal regulation highlights CES1 as a potential driver of metabolic programming during immune cell differentiation.

Metabolic impact of CES1 expression:

• Mitochondrial function:

  • CES1 supports maximal mitochondrial respiration capacity in DCs.

  • CES1 inhibition (via WWL113) impairs respiratory capacity .

  • This suggests CES1's involvement in maintaining oxidative metabolism in DCs.

• Glycolytic regulation:

  • CES1 inhibition increases lactate production, indicating a shift toward glycolysis .

  • This metabolic shift resembles the Warburg-like metabolism observed in activated immune cells.

• Metabolite homeostasis:

  • CES1-expressing DCs maintain higher levels of intracellular amino acids and TCA cycle intermediates .

  • This metabolic signature supports a more balanced metabolic state compared to the more inflammatory glycolytic state.

Functional consequences for immune responses:

• Maturation and activation markers:

  • CES1 inhibition leads to higher expression of CD209, CD86, and MHCII on DCs .

  • These surface markers are associated with enhanced antigen presentation and T cell stimulation capacity.

• Cytokine production:

  • CES1-inhibited DCs secrete higher quantities of pro-inflammatory cytokines (IL-6, IL-8, TNF) and also IL-10 .

  • This cytokine profile supports a more activated DC phenotype.

• Functional capabilities:

  • Enhanced phagocytic ability in CES1-inhibited DCs .

  • Increased capacity to polarize T helper 17 (Th17) differentiation in CES1-inhibited DCs .

  • These functional changes support CES1 as a regulator of DC effector functions.

In vivo relevance:
Transgenic overexpression of human CES1 in murine DCs resulted in:

• A less inflammatory DC phenotype .

• Increased resistance to T cell-mediated colitis .
  These findings establish CES1 as a potential therapeutic target in inflammatory conditions.

Mechanistic insights:
Transcriptomic analysis of CES1-inhibited DCs revealed:

• Enrichment of multiple inflammatory pathways .

• Altered metabolic gene expression profiles .
  This suggests that CES1 acts as a metabolic regulator that influences immune function through metabolic reprogramming.

Implications for immunometabolism research:

• Experimental design considerations:

  • Researchers should monitor CES1 expression when studying DC metabolism and function.

  • CES1 inhibitors (e.g., WWL113) can be used to manipulate DC metabolic state and function .

  • Genetic approaches (knockdown/overexpression) can further delineate CES1's role.

• Therapeutic potential:

  • CES1 modulation represents a novel approach to manipulate DC function for immunotherapy.

  • CES1 enhancement could potentially dampen inflammatory responses in autoimmune conditions.

  • CES1 inhibition might enhance vaccine efficacy by promoting more immunogenic DCs.

• Biomarker potential:

  • CES1 expression levels in DCs might serve as a biomarker for immunological states.

  • Monitoring CES1 activity could provide insights into metabolic reprogramming in immune cells.

This research establishes CES1 as a metabolic regulator that directs DC differentiation and function, with significant implications for our understanding of immunometabolism and potential therapeutic applications .

What are the species-specific differences in CES1 structure and function that researchers should consider in translational studies?

Understanding species-specific differences in CES1 structure and function is crucial for accurate interpretation and translation of research findings across species:

Molecular characteristics across species:

Functional divergences with research implications:

• Substrate specificity differences:

  • Human CES1 and rodent Ces1 can exhibit different affinities and rates of hydrolysis for the same substrates.

  • This can lead to significant differences in drug metabolism profiles between species.

  • Implication: Drug metabolism studies in rodents may not accurately predict human pharmacokinetics.

• Tissue distribution variations:

  • While liver expression is conserved across species, expression in immune cells shows species-specific patterns.

  • Human CES1 is highly expressed in monocytes/macrophages and shows specific regulation during dendritic cell differentiation .

  • Mouse Ces1 expression patterns in immune cells may differ, affecting inflammatory models.

  • Implication: Immune function studies should consider these distribution differences when translating findings.

• Genetic redundancy considerations:

  • Mice have multiple Ces1 genes (Ces1a-d) with overlapping functions.

  • Humans have a single CES1 gene with various polymorphisms.

  • Implication: Knockout studies in mice may require multiple gene targeting to fully eliminate Ces1 activity.

Antibody selection for cross-species studies:

• Species-specific antibodies:

  • Human-specific: MAB4920 (clone #544623) targets His19-Glu563 region .

  • Mouse-specific: AF7929 targets His19-Leu565 region of mouse CES1 .

• Multi-species reactive antibodies:

  • AF4920: Reacts with human, mouse, and rat CES1 .

  • Application recommendations: 0.2 μg/mL for Western blot of human/mouse/rat liver samples .

  • Expected detection: Human (63 kDa), mouse and rat (similar molecular weight) .

• Cross-reactivity validation:

  • When using antibodies across species, validation with positive controls from each species is essential.

  • The same antibody may require different dilutions for optimal detection across species.

Translational research considerations:

• Drug metabolism studies:

  • Humanized mouse models expressing human CES1 may provide more translatable results for pharmacokinetic studies.

  • In vitro studies using recombinant enzymes from different species can help identify species-specific metabolism patterns.

• Inflammatory and immune models:

  • CES1 overexpression in murine DCs generates a less inflammatory phenotype and increased resistance to T cell-mediated colitis .

  • This demonstrates that despite species differences, human CES1 can functionally impact mouse immune cells.

  • Studies should include both mouse and human cell experiments when possible for better translation.

• Antibody-based detection strategies:

  • For comparative studies across species, use antibodies validated for cross-reactivity (e.g., AF4920) .

  • Consider epitope conservation when selecting antibodies for evolutionary studies.

  • Validation data should be provided for each species under investigation.

Researchers conducting translational studies with CES1 should carefully consider these species-specific differences in experimental design, data interpretation, and when extrapolating findings across species .