pm20d1.2 Antibody

What is PM20D1 and why is it significant in metabolic research?

PM20D1 (peptidase M20 domain containing 1) is a secreted enzyme that functions as a bidirectional N-acyl amino acid synthase/hydrolase, regulating the endogenous N-fatty acyl amino acids (NAAs) in tissues and circulation . Its significance in metabolic research stems from its role in energy homeostasis and thermogenesis. PM20D1 is highly expressed in UCP1+ adipocytes (brown and beige fat cells) and is cold-inducible in subcutaneous white adipose tissue . The enzyme catalyzes both the condensation of fatty acids and amino acids to generate N-acyl amino acids and the reverse hydrolytic reaction. These N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration, thereby regulating energy expenditure . Research has demonstrated that increased circulating PM20D1 augments respiration and increases N-acyl amino acids in blood, suggesting its potential as a therapeutic target for treating obesity and associated metabolic disorders .

What are the structural characteristics of the PM20D1 protein?

PM20D1 is characterized by the presence of an M20 peptidase domain, which is shared among five members of the mammalian M20 peptidase family . The enzyme includes critical catalytic residues that coordinate divalent cations, specifically H125, D127, and H465, which are completely conserved between mouse and human PM20D1 . The human PM20D1 shares 71% identity and 86% similarity with the mouse enzyme . The protein possesses a signal peptide without any transmembrane domains, features characteristic of classically secreted proteins . PM20D1 has an observed molecular weight of approximately 55-60 kDa as detected in immunoblotting applications . The enzyme's three-dimensional structure enables its bidirectional catalytic function, allowing it to both synthesize and hydrolyze N-acyl amino acids with specificity for certain substrates .

What criteria should guide the selection of a PM20D1 antibody for specific research applications?

When selecting a PM20D1 antibody for specific research applications, researchers should consider several critical parameters:

• Application compatibility: Verify the antibody has been validated for your specific application (e.g., Western blot, ELISA, immunohistochemistry). The rabbit recombinant PM20D1 antibody (84397-2-PBS) has been validated for Western blot, Cytometric bead array, and Indirect ELISA applications .

• Species reactivity: Confirm cross-reactivity with your experimental species. The 84397-2-PBS antibody demonstrates validated reactivity with human and mouse samples .

• Clonality and host: Recombinant monoclonal antibodies like 84397-2-PBS provide unrivalled batch-to-batch consistency compared to polyclonal alternatives .

• Format compatibility: For multiplex assays, matched antibody pairs are essential. The 84397-2-PBS functions as a detection antibody paired with 84397-1-PBS as the capture antibody in Cytometric bead array applications .

• Buffer composition: For conjugation experiments, antibodies in PBS-only formulations (without BSA or azide) are required .

• Epitope location: Understanding the antibody's target region is crucial when studying specific PM20D1 domains or when potential post-translational modifications may affect recognition.

• Validation method: Review the validation data thoroughly, including positive and negative controls used, to ensure specificity for PM20D1.

What validation methods should be employed to confirm PM20D1 antibody specificity?

To rigorously validate PM20D1 antibody specificity, researchers should implement a multi-faceted approach:

• Knockout/knockdown controls: Test the antibody in tissues/cells with PM20D1 gene knockout or knockdown to confirm absence of signal.

• Overexpression controls: Evaluate antibody performance in cells overexpressing PM20D1 to verify increased signal intensity.

• Peptide competition assay: Pre-incubate the antibody with purified PM20D1 protein or immunizing peptide to demonstrate specific signal blocking.

• Western blot analysis: Confirm detection of a single band at the expected molecular weight (55-60 kDa for PM20D1) .

• Cross-species validation: Test reactivity across multiple species when conducting comparative studies.

• Mutant protein detection: Assess recognition of PM20D1 mutants, particularly those with mutations in catalytic residues (H125, D127, H465) to understand epitope location .

• Application-specific controls: For complex applications like Cytometric bead arrays, validate using matched antibody pairs with known positive samples .

• Comparative antibody analysis: Evaluate multiple antibodies targeting different PM20D1 epitopes to confirm consistent localization and expression patterns.

What are the advantages of using recombinant monoclonal antibodies for PM20D1 detection?

Recombinant monoclonal antibodies, such as the rabbit recombinant PM20D1 antibody (84397-2-PBS), offer several significant advantages for PM20D1 detection:

• Batch-to-batch consistency: Created using proprietary recombinant technology, these antibodies ensure unrivalled consistency between production lots, eliminating the variability inherent in traditional hybridoma-derived or polyclonal antibodies .

• Scalability: Recombinant production enables easy scale-up for larger experiments or long-term studies requiring consistent reagents .

• Supply security: The defined genetic sequence ensures future production capability and identical specificity regardless of production timing .

• Reduced background: Monoclonal antibodies typically produce cleaner results with less non-specific binding compared to polyclonal alternatives.

• Defined epitope recognition: Recombinant monoclonal antibodies target a single, specific epitope, enabling more precise monitoring of structural or post-translational modifications.

• Suitability for multiplexing: Their consistent performance makes recombinant monoclonal antibodies ideal for multiplex assays requiring matched pairs, mass cytometry, and multiplex imaging applications .

• Conjugation readiness: Formulations in PBS-only buffer (BSA and azide-free) make them immediately available for custom conjugation to various labels or substances .

What are the optimal conditions for Western blot detection of PM20D1?

For optimal Western blot detection of PM20D1, researchers should follow these methodological guidelines:

• Sample preparation:

  • For tissue samples, use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if investigating post-translational modifications

  • Maintain cold conditions throughout processing to prevent protein degradation

• Gel electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels for optimal resolution of PM20D1 (55-60 kDa)

  • Load 20-40 μg of total protein for cell/tissue lysates

  • Include positive controls (e.g., brown adipose tissue extracts)

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membrane (0.45 μm pore size) for better protein retention

• Blocking conditions:

  • 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • BSA blocking may provide better results for phospho-specific detection

• Primary antibody incubation:

  • Dilute rabbit recombinant PM20D1 antibody (84397-2-PBS) at 1:1000 to 1:2000

  • Incubate overnight at 4°C with gentle agitation

• Detection system:

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • For higher sensitivity, consider fluorescent secondary antibodies

• Controls:

  • Include positive control tissues known to express PM20D1 (brown adipose tissue, liver)

  • Consider knockout/knockdown controls to confirm specificity

• Expected results:

  • PM20D1 should appear as a band at approximately 55-60 kDa

  • Higher molecular weight bands may indicate post-translational modifications

How can the PM20D1 antibody be effectively utilized in multiplex assay systems?

The PM20D1 antibody can be effectively utilized in multiplex assay systems through the following methodological approaches:

• Cytometric bead array application:

  • Utilize the matched antibody pair: 84397-1-PBS as capture and 84397-2-PBS as detection antibodies

  • Conjugate the detection antibody (84397-2-PBS) with a fluorescent tag or biotin

  • Optimal working concentrations should be determined through titration experiments

  • Include appropriate isotype controls to assess non-specific binding

• Multiplex ELISA development:

  • Coat microplate wells with capture antibody (84397-1-PBS) at 1-10 μg/mL

  • Block with 1-5% BSA in PBS to minimize background

  • Use detection antibody (84397-2-PBS) at optimized concentration

  • Consider sandwich ELISA format for improved specificity

• Antibody conjugation for multiplex imaging:

  • The PBS-only formulation (BSA and azide-free) makes 84397-2-PBS ideal for direct conjugation

  • Compatible fluorophores include Alexa Fluor dyes, quantum dots, or metal isotopes for mass cytometry

  • Maintain a proper antibody:dye ratio to preserve binding capacity

  • Purify conjugated antibodies using size exclusion chromatography

• Mass cytometry applications:

  • Conjugate PM20D1 antibody with rare earth metals

  • Optimize staining concentration to maximize signal:noise ratio

  • Include appropriate biological controls for panel development

  • Analyze data using dimensionality reduction techniques (tSNE, UMAP)

• Multiplex tissue imaging:

  • For co-localization studies with other proteins of interest, use directly labeled antibodies

  • Optimize antibody concentration to minimize background

  • Sequential staining may be required if antibody species overlap

  • Include spectral unmixing controls when using fluorescent multiplexing

What methodological approaches enable precise quantification of PM20D1 enzymatic activity?

Precise quantification of PM20D1 enzymatic activity requires sophisticated methodological approaches that assess both its synthase and hydrolase functions:

• LC-MS/MS quantification of N-acyl amino acids:

  • Incubate purified PM20D1 with physiologically relevant concentrations of free oleate (300 μM) and phenylalanine (100 μM) in PBS at 37°C

  • Extract metabolites using liquid-liquid extraction with ethyl acetate or solid-phase extraction

  • Analyze using targeted multiple reaction monitoring (MRM) program

  • Quantify using isotopically labeled internal standards (e.g., C15-Phe)

  • For hydrolase activity, measure the release of free fatty acids from N-acyl amino acids

• Bidirectional activity assessment:

  • For synthase activity: Monitor formation of N-oleoyl phenylalanine (C18:1-Phe) when PM20D1 is incubated with oleate and phenylalanine

  • For hydrolase activity: Measure liberation of free oleate when PM20D1 is incubated with C18:1-Phe

  • Compare relative efficiency in both directions to understand enzymatic preference

• Substrate preference profiling:

  • Test various amino acids as amine head groups (phenylalanine shows highest efficiency)

  • Evaluate different fatty acids as acyl donors (free oleate, arachidonate, oleoyl-CoA)

  • Determine reaction kinetics for each substrate combination

• Mutational analysis to confirm activity:

  • Generate point mutations in catalytic residues (H125, D127, H465)

  • Compare wild-type and mutant enzyme activities under identical conditions

  • Ensures enzymatic activity is directly attributable to PM20D1 and not co-purifying proteins

• Activity preservation during purification:

  • Immunopurify Flag-tagged PM20D1 using magnetic Flag-M2 beads

  • Carefully titrate protein amounts for enzymatic assays

  • Maintain enzyme stability through appropriate buffer conditions

How can PM20D1 antibodies be utilized to investigate thermogenic activation in adipose tissues?

PM20D1 antibodies can be strategically employed to investigate thermogenic activation in adipose tissues through several advanced methodological approaches:

• Immunohistochemical co-localization studies:

  • Perform double immunostaining for PM20D1 and UCP1 to identify thermogenically active adipocytes

  • Quantify changes in PM20D1 expression in different adipose depots following cold exposure or β-adrenergic stimulation

  • Compare PM20D1 expression patterns between classical brown, beige, and white adipocytes

• Chromatin immunoprecipitation (ChIP) analysis:

  • Investigate transcriptional regulation of PM20D1 during thermogenic activation

  • Identify transcription factors binding to the PM20D1 promoter following cold exposure

  • Combine with PM20D1 antibody-based protein detection to correlate transcriptional changes with protein expression

• Secretion profile analysis:

  • Use PM20D1 antibodies in ELISA or Western blot to quantify PM20D1 secretion from adipose tissue explants

  • Compare secretion levels between adipose tissues from cold-exposed versus thermoneutral animals

  • Correlate PM20D1 secretion with N-acyl amino acid levels in conditioned media

• Flow cytometry of adipocyte populations:

  • Develop flow cytometry protocols using PM20D1 antibodies to identify and isolate PM20D1-expressing adipocytes

  • Characterize these populations for expression of other thermogenic markers

  • Perform single-cell transcriptomics on sorted populations to identify co-regulated genes

• In vivo imaging of thermogenic activation:

  • Conjugate PM20D1 antibodies with near-infrared fluorophores for non-invasive imaging

  • Monitor PM20D1 expression changes in vivo during cold adaptation

  • Correlate with metabolic parameters using indirect calorimetry

What approaches can resolve contradictory data regarding PM20D1 expression across different experimental systems?

Resolving contradictory data regarding PM20D1 expression across different experimental systems requires systematic investigation through multiple complementary approaches:

• Comprehensive antibody validation:

  • Validate antibody specificity using knockout controls across all experimental systems

  • Compare multiple antibodies targeting different PM20D1 epitopes

  • Develop rigorous positive and negative control panels specific to each experimental system

• Tissue-specific expression analysis:

  • Quantify PM20D1 mRNA and protein expression across multiple tissues (adipose depots, liver, kidney)

  • Account for potential tissue-specific post-translational modifications

  • Consider developmental timing and circadian variation in expression patterns

• Standardized isolation protocols:

  • Develop and adhere to standardized protocols for cell/tissue isolation

  • Document and control for variables that might affect PM20D1 expression (age, sex, diet, housing temperature)

  • Implement consistent protein extraction methods across laboratories

• Multi-omics integration:

  • Correlate transcriptomic, proteomic, and metabolomic data related to PM20D1

  • Identify potential discrepancies between mRNA and protein levels suggesting post-transcriptional regulation

  • Utilize data normalization techniques appropriate for cross-platform comparisons

• Genetic background considerations:

  • Evaluate PM20D1 expression across different mouse strains

  • Consider human population variations and their impact on expression patterns

  • Develop isogenic cell line models to control for genetic background effects

• Methodological documentation:

  • Thoroughly document experimental conditions, including housing temperature, which significantly affects thermogenic gene expression

  • Report precise antibody dilutions, incubation times, and detection systems used

  • Consider creating a standardized reporting format for PM20D1 studies to facilitate cross-laboratory comparisons

How can researchers investigate the relationship between PM20D1 activity and mitochondrial function?

Investigating the relationship between PM20D1 activity and mitochondrial function requires sophisticated experimental approaches that bridge enzymatic activity with functional mitochondrial outcomes:

• Oxygen consumption measurements:

  • Perform respirometry on isolated mitochondria treated with N-acyl amino acids (PM20D1 products)

  • Measure oxygen consumption rates in cells with modulated PM20D1 expression

  • Assess both basal and maximal respiration, as well as coupling efficiency

• Mitochondrial binding assays:

  • Use fluorescently labeled N-acyl amino acids to visualize direct binding to mitochondria

  • Perform mitochondrial isolation followed by binding studies with purified N-acyl amino acids

  • Identify specific mitochondrial binding partners for N-acyl amino acids using crosslinking approaches

• UCP1-dependent versus independent thermogenesis:

  • Compare mitochondrial responses in wild-type versus UCP1-knockout models

  • Assess PM20D1-mediated effects on energy expenditure in both genetic backgrounds

  • Quantify N-acyl amino acid levels and correlate with thermogenic capacity

• Mitochondrial membrane potential analysis:

  • Measure changes in membrane potential using potential-sensitive dyes

  • Investigate how PM20D1-generated N-acyl amino acids affect proton leak

  • Compare with known uncouplers to characterize mechanism of action

• Metabolic flux analysis:

  • Track carbon flux through mitochondrial metabolic pathways in cells with altered PM20D1 activity

  • Measure changes in TCA cycle intermediates and correlate with N-acyl amino acid levels

  • Assess fatty acid oxidation rates in response to PM20D1 modulation

• In vivo metabolic phenotyping:

  • Measure energy expenditure in mice with altered PM20D1 expression using indirect calorimetry

  • Track body temperature during cold challenge in PM20D1-overexpressing models

  • Correlate circulating N-acyl amino acid levels with whole-body energy expenditure

What methodological approaches can differentiate between the synthase and hydrolase activities of PM20D1?

Differentiating between the synthase and hydrolase activities of PM20D1 requires tailored experimental approaches that selectively measure each direction of this bidirectional enzyme:

• Optimized reaction conditions:

  • For synthase activity: Incubate PM20D1 with oleate (1.5 mM) and phenylalanine (0.1 mM) in PBS at 37°C

  • For hydrolase activity: Incubate PM20D1 with N-oleoyl phenylalanine in PBS at 37°C

  • Track reaction progress over time to establish kinetic parameters for each direction

• LC-MS/MS detection methods:

  • Develop specific MRM transitions for both substrates and products

  • For synthase activity: Monitor formation of N-oleoyl phenylalanine (m/z = 428)

  • For hydrolase activity: Monitor release of free oleate from N-acyl amino acids

  • Use isotopically labeled internal standards for accurate quantification

• Substrate concentration effects:

  • Evaluate directional preference by varying substrate concentrations

  • Determine if the preferred direction changes under different physiological conditions

  • Calculate kinetic parameters (Km, Vmax) for both directions to understand enzymatic efficiency

• pH dependence analysis:

  • Measure both activities across a range of pH values (5.0-9.0)

  • Determine if synthase and hydrolase activities have different pH optima

  • Correlate with potential subcellular localization environments

• Point mutation effects:

  • Examine if specific mutations differentially affect synthase versus hydrolase activity

  • Engineer mutations that preferentially impact one activity over the other

  • Use these findings to understand the catalytic mechanism

• Compartmentalized cellular assays:

  • Develop subcellular targeting constructs to localize PM20D1 to specific organelles

  • Assess if localization affects directional preference of enzymatic activity

  • Correlate with natural enzyme distribution in cells

What are the most effective methods for quantifying N-acyl amino acids in biological samples?

Effective quantification of N-acyl amino acids in biological samples requires sophisticated analytical approaches that ensure both sensitivity and specificity:

• Sample preparation optimization:

  • For plasma: Protein precipitation with methanol followed by liquid-liquid extraction

  • For tissues: Homogenization in methanol/chloroform mixtures (2:1 v/v)

  • Include antioxidants (BHT) to prevent oxidation of unsaturated fatty acid moieties

  • Use internal standards (e.g., C15-Phe) added at the beginning of extraction

• Chromatographic separation techniques:

  • Reversed-phase HPLC with C18 columns for optimal separation

  • Mobile phase gradient from aqueous (with 0.1% formic acid) to organic (acetonitrile/methanol)

  • Temperature-controlled columns (40°C) for reproducible retention times

  • Nano-LC approaches for enhanced sensitivity with limited sample amounts

• Mass spectrometry detection methods:

  • Targeted multiple reaction monitoring (MRM) for quantification of known N-acyl amino acids

  • High-resolution MS for untargeted discovery of novel N-acyl amino acid species

  • Characteristic fragmentation patterns: N-oleoyl phenylalanine yields m/z = 164 product ion (phenylalanine anion)

  • ESI negative mode ionization for optimal sensitivity

• Quantification strategies:

  • Absolute quantification using isotopically labeled internal standards

  • Baseline plasma concentrations of specific N-acyl amino acids range from 1-100 nM

  • Multi-point calibration curves with matrix-matched standards

  • Quality control samples to monitor extraction efficiency and instrument performance

• Comprehensive metabolite coverage:

  • Monitor various combinations of fatty acids (C14-C22) and amino acids

  • Include both saturated and unsaturated fatty acid conjugates

  • Focus on physiologically relevant N-acyl amino acids (C18:1-Phe, C18:1-Leu/Ile)

  • Consider measurement of both free fatty acids and amino acids to understand substrate availability

How can researchers distinguish PM20D1 activity from other enzymes affecting N-acyl amino acid metabolism?

Distinguishing PM20D1 activity from other enzymes affecting N-acyl amino acid metabolism requires careful experimental design and controls:

• Selective inhibitor profiling:

  • Develop and validate selective inhibitors of PM20D1

  • Test against related enzymes in the M20 peptidase family

  • Determine if inhibition patterns match expected PM20D1 activity profiles

  • Use established inhibitors of related enzymes (e.g., FAAH inhibitors) as controls

• Substrate specificity analysis:

  • PM20D1 shows preference for free fatty acids over acyl-CoA derivatives

  • PM20D1 efficiently uses phenylalanine as amine head group

  • PM20D1 does not hydrolyze N-oleoyl ethanolamine (C18:1-EA), distinguishing it from FAAH

  • Use these distinctive substrate preferences to differentiate enzymatic sources

• Genetic manipulation approaches:

  • Create PM20D1 knockout models and measure residual N-acyl amino acid metabolism

  • Perform selective knockdown of PM20D1 versus other candidate enzymes

  • Rescue experiments with wild-type versus catalytically inactive PM20D1 mutants

  • Combine with metabolomic profiling to identify PM20D1-specific metabolite signatures

• Tissue expression correlation studies:

  • Compare tissue distribution of PM20D1 with other candidate enzymes

  • Correlate expression levels with N-acyl amino acid profiles across tissues

  • Identify tissues where PM20D1 is the predominant enzyme affecting these metabolites

• In vitro reconstitution experiments:

  • Purify recombinant enzymes and compare their activities directly

  • Test with various substrate combinations to identify unique activity signatures

  • Perform competition experiments to assess relative contributions

• Point mutant comparative analysis:

  • Create point mutations in catalytic residues specific to PM20D1 (H125, D127, H465)

  • Compare with mutations in homologous residues of related enzymes

  • Use to establish structure-function relationships unique to PM20D1

What bioinformatic approaches can integrate PM20D1 expression data with broader metabolic networks?

Integrating PM20D1 expression data with broader metabolic networks requires sophisticated bioinformatic approaches that bridge transcriptomics, proteomics, and metabolomics:

• Co-expression network analysis:

  • Construct networks of genes co-regulated with PM20D1 across tissues and conditions

  • Identify hub genes and potential transcriptional regulators

  • Apply weighted gene correlation network analysis (WGCNA) to identify modules of functionally related genes

  • Correlate modules with physiological traits (e.g., energy expenditure, cold tolerance)

• Pathway enrichment analysis:

  • Map PM20D1 and associated genes to canonical metabolic pathways

  • Identify significantly enriched pathways using tools like KEGG, Reactome, or MetaboAnalyst

  • Perform gene set enrichment analysis (GSEA) using custom gene sets related to thermogenesis

  • Integrate with metabolomic data to identify affected metabolic fluxes

• Multi-omics data integration:

  • Correlate PM20D1 mRNA expression with protein levels and N-acyl amino acid concentrations

  • Apply multi-block partial least squares (MBPLS) to integrate heterogeneous data types

  • Develop Bayesian networks to infer causal relationships between PM20D1 activity and downstream metabolic effects

  • Use similarity network fusion (SNF) to identify patterns across different data types

• Metabolic flux analysis:

  • Develop constraint-based metabolic models incorporating PM20D1 activity

  • Simulate flux distributions under various conditions (e.g., cold exposure, fed vs. fasted)

  • Predict metabolic consequences of PM20D1 modulation

  • Validate predictions with experimental isotope tracing data

• Cross-species comparative analysis:

  • Compare PM20D1 sequence, structure, and function across species

  • Identify evolutionarily conserved regulatory elements in the PM20D1 gene

  • Correlate species-specific PM20D1 activity with metabolic adaptations (e.g., hibernation, cold adaptation)

  • Apply phylogenetic approaches to understand evolutionary constraints on PM20D1 function

How can mass spectrometry techniques be optimized for comprehensive profiling of PM20D1 substrates and products?

Optimizing mass spectrometry techniques for comprehensive profiling of PM20D1 substrates and products requires sophisticated methodological refinements:

• Sample preparation enhancement:

  • Develop selective extraction methods for N-acyl amino acids

  • Implement differential extraction protocols to separate substrates from products

  • Include sequential extraction steps to capture compounds with varying polarities

  • Minimize degradation through rapid processing and appropriate stabilizers

• Advanced LC separation strategies:

  • Implement 2D-LC approaches for improved separation of complex matrices

  • Utilize HILIC chromatography for polar metabolites and reverse-phase for lipid components

  • Optimize mobile phase composition for maximum sensitivity

  • Develop specialized gradients for isomer separation (e.g., leucine vs. isoleucine conjugates)

• High-resolution mass spectrometry applications:

  • Employ quadrupole-orbitrap or Q-TOF instruments for accurate mass determination

  • Implement data-dependent acquisition for untargeted discovery

  • Utilize parallel reaction monitoring (PRM) for targeted quantification with high specificity

  • Apply ion mobility separation for additional compound differentiation

• Comprehensive substrate/product coverage:

  • Develop targeted methods for all potential N-acyl amino acid combinations

  • Include various fatty acid chain lengths (C14-C22) and saturation states

  • Monitor all 20 proteinogenic amino acids as potential amine donors

  • Establish detection parameters for both high and low abundance compounds

• Isotope labeling strategies:

  • Implement stable isotope labeling to track PM20D1-specific transformations

  • Design pulse-chase experiments to determine flux through PM20D1 pathways

  • Apply isotopomer analysis to distinguish enzymatic from non-enzymatic reactions

  • Develop metabolic flux analysis integrating PM20D1 activity

• Integrated data processing pipelines:

  • Implement automated peak detection and alignment algorithms

  • Develop custom libraries of N-acyl amino acid spectra

  • Apply machine learning for improved compound identification

  • Integrate with metabolic pathway databases for biological context

What are the best experimental designs for investigating PM20D1's role in UCP1-independent thermogenesis?

Investigating PM20D1's role in UCP1-independent thermogenesis requires carefully designed experimental approaches that differentiate between multiple thermogenic mechanisms:

• Genetic model systems:

  • Utilize UCP1 knockout mice to isolate UCP1-independent thermogenic mechanisms

  • Develop PM20D1 tissue-specific knockout and overexpression models

  • Create double knockout models (UCP1/PM20D1) to assess additive or synergistic effects

  • Engineer models with constitutively active or inactive PM20D1 to assess enzymatic contribution

• Comprehensive metabolic phenotyping:

  • Perform indirect calorimetry in various housing temperatures (thermoneutrality vs. cold)

  • Measure whole-body O₂ consumption and CO₂ production with high temporal resolution

  • Assess acute responses to β-adrenergic stimulation

  • Quantify cold tolerance and adaptation through core temperature monitoring

• Cellular bioenergetics assessment:

  • Measure respiration in primary adipocytes with modulated PM20D1 activity

  • Assess the effects of specific N-acyl amino acids on mitochondrial function

  • Quantify mitochondrial membrane potential in response to N-acyl amino acid treatment

  • Compare respiratory responses in brown, beige, and white adipocytes

• Molecular pathway interrogation:

  • Investigate if PM20D1 activity intersects with other UCP1-independent thermogenic mechanisms (creatine cycle, calcium cycling)

  • Determine if N-acyl amino acids directly interact with mitochondrial proteins or membranes

  • Assess if PM20D1-dependent pathways are regulated by classic thermogenic stimuli (cold, β-adrenergic signaling)

  • Identify potential target pathways through transcriptomic analysis of tissues from PM20D1-modulated animals

• Translational human studies:

  • Correlate circulating PM20D1 and N-acyl amino acid levels with metabolic parameters in humans

  • Investigate associations between PM20D1 genetic variants and thermal regulation

  • Perform cold exposure studies in humans with metabolomic profiling of N-acyl amino acids

  • Assess PM20D1 expression in human adipose tissue biopsies from different depots