Recombinant Mouse All-trans-retinol 13,14-reductase (Retsat)

What is the primary enzymatic function of Retsat and how is it typically measured in vitro?

Retsat (retinol saturase) is an NADH/NADPH-dependent oxidoreductase that specifically saturates the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol (all-trans-DROL). This enzymatic activity is substrate-specific, as Retsat does not use all-trans-retinoic acid nor 9-cis, 11-cis, or 13-cis-retinol isomers as substrates .

For in vitro measurement of Retsat activity, researchers typically employ:

• Enzyme assays using recombinant purified Retsat protein with all-trans-retinol as substrate

• HPLC analysis to separate and quantify the conversion of all-trans-retinol to all-trans-13,14-dihydroretinol

• LC-MS/MS methods for more sensitive detection of dihydroretinoid metabolites

• Spectrophotometric assays measuring NAD(P)H consumption during the reaction

The enzyme contains a critical FAD/NAD dinucleotide-binding motif essential for its catalytic activity, and mutation of this motif abolishes enzymatic function .

How does Retsat expression vary across different mouse tissues and under various physiological conditions?

Retsat shows a tissue-specific expression pattern with significant regulation under various physiological conditions:

Tissue distribution:

• Highest expression: liver, kidney, adipose tissue, and intestine

• Moderate expression: brain, heart, and lung

• Within adipose tissue: expressed predominantly in adipocytes rather than stromal vascular fraction

Physiological regulation:

• Cold exposure: Significantly upregulated in brown adipose tissue (BAT) and white adipose tissue (WAT)

• β-adrenergic stimulation: Strongly induced in adipocytes through β-adrenergic signaling pathways

• Obesity: Downregulated in white adipose tissue in both diet-induced and genetic obesity models

• Thiazolidinedione treatment: Increases Retsat expression in adipose tissue of obese mice

Quantification methods include qRT-PCR for mRNA expression, Western blotting for protein levels, and ELISA for precise protein quantification in tissue homogenates and cell lysates .

What experimental models are most suitable for studying Retsat function in adipogenesis?

Several experimental models have proven effective for investigating Retsat's role in adipogenesis:

In vitro models:

• 3T3-L1 preadipocyte differentiation: Standard model where Retsat expression increases during differentiation

• Primary mouse embryonic fibroblasts (MEFs): Can be differentiated into adipocytes and allow for genetic manipulation

• Brown adipocyte cell lines: Useful for studying Retsat's role in thermogenic adipocytes

Genetic manipulation approaches:

• siRNA or shRNA knockdown: For acute depletion of Retsat

• CRISPR/Cas9 gene editing: For complete knockout or introducing specific mutations (e.g., in the FAD/NAD binding motif)

• Retroviral/lentiviral overexpression: For gain-of-function studies

Key experimental findings:

• Ablation of Retsat dramatically inhibits adipogenesis

• Ectopic expression of Retsat with intact FAD/NAD dinucleotide-binding motif promotes adipogenesis

• Retsat is not required for adipogenesis when exogenous PPARγ ligands are provided

When designing experiments, researchers should consider that Retsat appears to have functions beyond its enzymatic activity, as supplementation with dihydroretinol (the product of Retsat) fails to rescue the adipogenic defect in Retsat-depleted cells .

What are the recommended methods for purifying and characterizing recombinant mouse Retsat protein?

For optimal purification and characterization of recombinant mouse Retsat:

Expression systems:

• E. coli: For high yield but may require refolding due to membrane association

• Insect cells (Sf9, High Five): Better for obtaining properly folded protein with post-translational modifications

• Mammalian cells (HEK293, CHO): For most native-like protein structure and modifications

Purification strategy:

• Add an N-terminal tag (His, GST, or FLAG) after the ER signal peptide to avoid interference with localization

• Use detergent solubilization (e.g., CHAPS, DDM) since Retsat is an ER membrane-associated protein

• Employ affinity chromatography followed by size exclusion chromatography for higher purity

Characterization methods:

• Enzymatic activity assay: Using all-trans-retinol as substrate and monitoring product formation by HPLC

• Binding assays: For cofactor (NAD/NADPH) and substrate binding analyses

• Circular dichroism: To assess secondary structure integrity

• Thermal shift assay: To determine protein stability under various conditions

Critical considerations:

• Include FAD/NAD in buffers to stabilize the enzyme

• Store with reducing agents to prevent oxidation of critical cysteine residues

• Consider using recombinant fragments (like aa 333-451) as controls for antibody validation

How do mutations in the FAD/NAD dinucleotide-binding motif affect Retsat's functionality?

Mutations in the FAD/NAD dinucleotide-binding motif have profound effects on Retsat function:

Structural implications:

• The FAD/NAD binding motif is essential for cofactor binding and catalytic activity

• In recent computational studies, NADH was identified as the optimal cofactor for Retsat

Functional consequences of mutations:

• Complete loss of enzymatic activity: Mutated Retsat fails to convert all-trans-retinol to all-trans-13,14-dihydroretinol

• Abolished promotion of adipogenesis: Unlike wild-type Retsat, the mutated form cannot enhance adipocyte differentiation

• Reduced PPARγ activation: Mutations prevent Retsat from increasing endogenous PPARγ transcriptional activity

Naturally occurring mutations:

• The Q247R mutation, observed in Qinghai-Tibet Plateau mammals as an adaptation to hypoxia, shows altered functionality

• This mutation inhibits tumor growth in vivo and may represent a gain-of-function mutation

Researchers can engineer specific mutations in the FAD/NAD binding motif using site-directed mutagenesis to investigate structure-function relationships. When expressed in cells, these mutants can be compared to wild-type Retsat for enzymatic activity, subcellular localization, and effects on downstream pathways .

What methodologies are most effective for studying Retsat's role in thermogenesis?

To investigate Retsat's function in thermogenesis, researchers can employ these methodologies:

In vivo approaches:

• BAT-specific Retsat knockout mice: Generated using Cre-lox system with UCP1-Cre or similar BAT-specific promoters

• Cold exposure experiments: Testing acute (4-6 hours) and chronic (days to weeks) cold tolerance at 4°C

• Metabolic phenotyping: Measuring oxygen consumption, CO2 production, and heat generation in metabolic chambers

• Norepinephrine or CL-316,243 (β3-adrenergic agonist) injection: To assess β-adrenergic response

Ex vivo and in vitro methods:

• Primary brown adipocyte isolation and differentiation: From interscapular BAT

• Oxygen consumption measurements: Using Seahorse XF analyzers to assess mitochondrial respiration

• Lipolysis assays: Measuring glycerol and free fatty acid release upon β-adrenergic stimulation

• Thermogenic gene expression analysis: qRT-PCR for UCP1, PGC1α, PRDM16, etc.

Key experimental findings:

• RetSat expression is induced by β-adrenergic signaling and cold exposure

• Loss of RetSat in brown adipocytes reduces thermogenic gene expression

• Mice lacking RetSat in BAT have impaired acute cold tolerance

• RetSat depletion interferes with lipolysis in both brown and white adipocytes

These methodologies allow for comprehensive evaluation of Retsat's role in adaptive thermogenesis from molecular to organismal levels.

How can researchers differentiate between Retsat's enzymatic and non-enzymatic functions in cellular models?

Distinguishing between enzymatic and non-enzymatic functions of Retsat requires specialized experimental approaches:

Experimental strategies:

• Enzyme-dead mutants:

  • Generate Retsat with mutations in the FAD/NAD binding motif that abolish enzymatic activity but maintain protein structure

  • Compare effects of wild-type vs. enzyme-dead Retsat on cellular phenotypes

• Metabolite supplementation experiments:

  • Add all-trans-13,14-dihydroretinol (Retsat's enzymatic product) to Retsat-depleted cells

  • Phenotypes rescued by dihydroretinol are likely dependent on enzymatic activity

  • Notably, supplementation with dihydroretinol failed to rescue adipogenesis in Retsat-depleted cells, suggesting non-enzymatic functions

• Protein-protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry to identify Retsat-interacting proteins

  • Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to Retsat

  • The STRING database has identified Pin1 and Akt1 as potential Retsat-interacting proteins

• Subcellular localization experiments:

  • Fluorescent tagging and microscopy to determine if enzymatic activity affects localization

  • Subcellular fractionation to isolate ER-associated vs. other cellular pools of Retsat

Research findings showing dual functions:

• Retsat's role in PPARγ activation appears independent of its enzymatic product

• Retsat's function in ferroptosis involves both enzymatic activity (converting retinol to less protective dihydroretinol) and potential non-enzymatic effects on lipid metabolism

What is the current understanding of Retsat's role in cancer biology and how can it be investigated?

Retsat demonstrates complex roles in cancer biology that can be investigated through several approaches:

Current understanding:

• Retsat is mostly downregulated in multiple types of human cancers

• Lower Retsat expression correlates with worse clinical outcomes in skin cutaneous melanoma (SKCM)

• The Q247R mutation in Retsat inhibits tumor growth in vivo

• Methylation of the Retsat promoter is increased in cancer tissues

• Retsat expression positively correlates with immune cell infiltration in tumors

Proposed mechanisms:

• Inhibition of Pin1-related signaling pathway

• Reduction of phosphorylated Akt1 levels

• Regulation of ferroptosis through retinoid metabolism

Investigation methodologies:

• Expression analysis in human cancers:

  • Mining TCGA, GEO, and other public databases

  • IHC staining of tissue microarrays

  • qRT-PCR and Western blot of tumor vs. normal tissues

• Functional studies:

  • CRISPR/Cas9 knockout or overexpression in cancer cell lines

  • Xenograft tumor models with Retsat-modified cells

  • DMBA/TPA-induced skin carcinogenesis in Retsat mutant mice

• Epigenetic regulation:

  • Bisulfite sequencing of Retsat promoter region

  • ChIP assays to identify transcription factors binding to Retsat promoter

  • Treatment with DNA methyltransferase inhibitors (e.g., 5-azacytidine)

• Signaling pathway analysis:

  • Western blot for Pin1, phospho-Akt, and other signaling molecules

  • Co-immunoprecipitation to detect protein-protein interactions

  • Luciferase reporter assays for relevant pathways (PI3K/Akt)

How does Retsat contribute to ferroptosis regulation and what experimental approaches can elucidate this function?

Retsat plays a significant role in ferroptosis regulation through retinoid metabolism:

Mechanistic insights:

• Retsat promotes ferroptosis by transforming retinol to 13,14-dihydroretinol

• This conversion changes a strong anti-ferroptosis regulator (retinol) into a relatively weak one (dihydroretinol)

• Retinoids (retinol, retinal, retinoic acid) act as radical-trapping antioxidants that protect lipid membranes

• Additionally, retinoic acid activates stearoyl-CoA desaturase transcription via nuclear receptors, promoting monounsaturated fatty acid production and ferroptosis resistance

Experimental approaches to study Retsat in ferroptosis:

• Cell viability assays:

  • Treating Retsat-depleted vs. control cells with ferroptosis inducers (erastin, RSL3, etc.)

  • Rescue experiments with ferroptosis inhibitors (ferrostatin-1, liproxstatin-1)

  • Supplementation with retinoids or dihydroretinoids to assess their protective effects

• Lipid peroxidation measurements:

  • C11-BODIPY or PEROX-H2 fluorescent probes for live-cell imaging

  • MDA or 4-HNE assays for fixed endpoint measurements

  • Lipidomics to profile oxidized phospholipid species

• Mechanistic studies:

  • Gene expression analysis of stearoyl-CoA desaturase and other lipid metabolism genes

  • ChIP assays to assess retinoic acid receptor binding to target promoters

  • Pseudotargeted lipidomic analysis to identify associations between retinoid regulation and lipid metabolism

• Retinoid metabolism tracking:

  • LC-MS/MS to quantify retinol, dihydroretinol, retinoic acid, and dihydroretinoic acid

  • Isotope-labeled retinol tracing to follow metabolic conversion rates

For researchers entering this field, combining genetic manipulation of Retsat with comprehensive ferroptosis phenotyping and metabolite analysis offers the most thorough approach to understanding its role in this cell death pathway.

What approaches can be used to investigate the relationship between Retsat and PPARγ signaling?

The relationship between Retsat and PPARγ signaling can be studied through several complementary approaches:

Established relationship:

• RetSat is directly regulated by PPARγ as a transcriptional target

• RetSat with intact FAD/NAD dinucleotide-binding motif increases endogenous PPARγ transcriptional activity

• RetSat is not required for adipogenesis when cells are provided with exogenous PPARγ ligands

• RetSat expression in adipose tissue is increased by thiazolidinedione treatment (PPARγ agonists)

Investigation methodologies:

• Transcriptional regulation:

  • ChIP assays to confirm PPARγ binding to RetSat promoter

  • Luciferase reporter assays with wild-type and mutated RetSat promoter constructs

  • Analysis of RetSat expression after treatment with various PPARγ agonists/antagonists

• RetSat effects on PPARγ activity:

  • PPARγ-responsive element (PPRE) luciferase reporter assays in cells with:

    • RetSat overexpression (wild-type vs. enzyme-dead mutants)

    • RetSat knockdown or knockout

  • qRT-PCR of PPARγ target genes (e.g., aP2, CD36, adiponectin)

  • Co-immunoprecipitation to detect potential physical interactions

• Metabolite studies:

  • Determine if RetSat-generated metabolites activate PPARγ using:

    • Ligand binding assays with purified PPARγ ligand-binding domain

    • Competitive displacement assays with known PPARγ ligands

    • Structural studies (e.g., X-ray crystallography) of PPARγ with RetSat metabolites

• Functional studies in adipocytes:

  • Comparison of adipogenesis in:

    • Wild-type cells

    • RetSat-depleted cells

    • RetSat-depleted cells supplemented with PPARγ ligands

    • RetSat-depleted cells supplemented with all-trans-13,14-dihydroretinol

These approaches can help elucidate whether RetSat's effects on PPARγ signaling are mediated through its enzymatic activity, protein-protein interactions, or other mechanisms, providing crucial insights into adipocyte differentiation and function.