GSTP1 Mouse

What is GSTP1 and what are its primary functions in mice?

GSTP1 is an isozyme of the glutathione S-transferase family abundantly expressed in various mouse tissues. It serves dual functions in mice:

• Detoxification: GSTP1 catalyzes the conjugation of glutathione to electrophilic compounds, facilitating their elimination.

• Signaling regulation: GSTP1 functions as an inhibitor of JNK and influences ERK1/ERK2 pathways, thereby regulating cellular stress responses and proliferation.

Mouse embryo fibroblasts (MEFs) from GSTP1 knockout mice (GSTpi(-/-)) demonstrate significantly faster population doubling (26.2 hours vs. 33.6 hours in wild-type), indicating that GSTP1 normally moderates cell proliferation . Additionally, when GSTP1 is reintroduced via retroviral transfection into knockout cells, the doubling time increases to 30.4 hours, further supporting its regulatory role in proliferation .

How does mouse GSTP1 structure compare to human GSTP1?

Mouse GSTP1 shares significant structural similarities with human GSTP1:

• Both exist in monomeric and dimeric forms, with monomers having a molecular mass of approximately 23.3 kDa and dimers approximately 46.5 kDa .

• The monomeric form appears to function as a JNK inhibitor in both species .

• Both human and mouse GSTP1 demonstrate heterogeneity in subunit composition due to post-translational modifications .

• The C-terminal portion of both mouse and human GSTP1 serves as the binding region for JNK .

MALDI-TOF mass spectrometry analysis reveals that mouse GSTP1 monomers, like human GSTP1, exhibit heterogeneity with secondary peaks differing from primary peaks by approximately 200-210 atomic mass units, suggesting similar patterns of post-translational modifications across species .

What are the expression patterns of GSTP1 in normal mouse tissues?

GSTP1 expression in mice shows distinct tissue and developmental patterns:

• Developmental regulation: GSTP1 is expressed as a fetal enzyme in mouse hepatocytes but is typically absent in adult, differentiated hepatocytes .

• Tissue distribution: GSTP1 is abundantly expressed in non-hepatic tissues including lung, kidney, intestine, and certain immune cells.

• Stress induction: GSTP1 expression can be induced in various tissues in response to oxidative stress and xenobiotic exposure.

• Reexpression in pathology: GSTP1 is often reexpressed in liver during regeneration, chronic inflammation, and carcinogenesis .

This expression pattern makes GSTP1 a useful marker for de-differentiation in various mouse models of disease, particularly in cancer studies where increased GSTP1 expression often correlates with malignant transformation .

What are the key phenotypic differences between GSTP1 knockout and wild-type mice?

GSTP1 knockout mice (GSTpi(-/-)) exhibit several distinctive phenotypes compared to wild-type (GSTpi(+/+)) mice:

• Accelerated cell proliferation: MEFs from knockout mice show faster population doubling times (26.2 hours vs. 33.6 hours in wild-type) .

• Altered hematopoiesis: Knockout mice have higher basal levels of circulating white blood cells .

• Enhanced ERK1/ERK2 signaling: Both early passage and immortalized MEFs from knockout mice show significantly elevated ERK1/ERK2 activity .

• Normal development: Despite these cellular differences, knockout mice develop normally with no obvious physical abnormalities under standard conditions .

• Altered drug responses: Knockout mice do not respond to GSTP1 inhibitors like TLK199, which stimulates lymphocyte production and bone marrow progenitor proliferation only in wild-type mice .

How does GSTP1 deficiency affect stress responses in mouse models?

GSTP1 knockout mice demonstrate altered cellular responses to various stressors:

• Enhanced JNK activation: Without GSTP1's inhibitory effect, JNK activation is prolonged and amplified under stress conditions .

• Altered oxidative stress handling: Knockout mice show modified responses to oxidative challenges due to both the loss of GSTP1's catalytic detoxification function and its regulatory role in stress-response signaling .

• Cell survival impact: MEFs from knockout mice show different patterns of apoptosis in response to stress signals, with stress conditions that induce high levels of apoptosis in wild-type cells sometimes allowing continued proliferation in knockout-derived cells .

• Compensatory mechanisms: Extended exposure to stressors can trigger upregulation of other detoxification enzymes in knockout mice, representing adaptive responses .

These altered stress responses make GSTP1 knockout mice valuable models for studying cellular adaptation to various environmental challenges and for investigating the role of GSTP1 in disease processes involving oxidative stress.

What methodological considerations are important when generating and validating GSTP1 knockout mice?

When generating and validating GSTP1 knockout models, researchers should consider:

• Knockout strategy selection:

  • Traditional homologous recombination in embryonic stem cells

  • CRISPR/Cas9-mediated gene editing for precise modifications

  • Conditional knockout systems using Cre-loxP for tissue-specific or inducible deletion

• Comprehensive validation approaches:

  • Genotyping PCR with primers spanning the targeted region

  • Western blot analysis to confirm complete absence of GSTP1 protein

  • Enzymatic activity assays using CDNB (1-chloro-2,4-dinitrobenzene) to verify functional knockout

  • RT-PCR to confirm absence of GSTP1 mRNA transcripts

• Control selection:

  • Littermate controls are preferred to account for genetic background effects

  • Heterozygous mice should be included to assess gene dosage effects

  • Wild-type controls from the same colony to minimize environmental variables

• Phenotypic characterization:

  • Baseline hematological parameters, including white blood cell counts

  • MEF proliferation rates under standard conditions

  • ERK1/ERK2 activation status in various tissues and isolated cells

  • Response to GSTP1 inhibitors like TLK199

How does GSTP1 regulate JNK signaling in mouse cells?

GSTP1 regulates JNK signaling through direct protein-protein interactions:

• Binding mechanism: Monomeric GSTP1 appears to be the primary form that binds to JNK, serving as an inhibitor under normal conditions .

• Binding region: The C-terminal portion of GSTP1 is critical for JNK binding in mouse cells, similar to the interaction observed in human cells .

• Stress-induced dissociation: Under stress conditions, GSTP1 dissociates from JNK, allowing JNK activation and subsequent phosphorylation of downstream targets.

• Regulatory phosphorylation: Phosphorylation of GSTP1 at serine residues in the C-terminal portion where JNK binds may modulate this interaction .

To study this interaction, researchers can employ:

• Co-immunoprecipitation assays with anti-GSTP1 or anti-JNK antibodies

• Proximity ligation assays for in situ detection of protein-protein interactions

• FRET/BRET approaches using fluorescently labeled proteins to monitor interactions in living cells

• In vitro binding assays with purified components to determine binding kinetics

What is the relationship between GSTP1 and ERK1/ERK2 signaling in mice?

GSTP1 influences ERK1/ERK2 signaling pathways:

• Enhanced ERK activity in GSTP1-deficient cells: MEFs from GSTpi(-/-) mice exhibit significantly elevated ERK1/ERK2 activity compared to wild-type cells .

• Proliferation effects: The increased ERK activity in knockout cells correlates with accelerated proliferation rates, suggesting GSTP1 normally moderates ERK-dependent proliferative signals .

• Regulatory mechanism: Unlike its direct interaction with JNK, GSTP1 may regulate ERK pathways indirectly, possibly through modulation of upstream kinases or through cross-talk with the JNK pathway.

• Pathway integration: GSTP1 appears to integrate multiple signaling pathways, forming part of a regulatory network that balances proliferation with stress responses.

Experimental approaches to study GSTP1-ERK relationships include:

• Western blot analysis of ERK phosphorylation status in various tissues from wild-type and knockout mice

• ERK activity assays using specific substrates

• Pharmacological manipulation with ERK pathway inhibitors

• Analysis of ERK nuclear translocation and target gene activation

How do post-translational modifications of GSTP1 affect its signaling functions in mice?

GSTP1 undergoes several post-translational modifications that influence its signaling functions:

• Phosphorylation: LC-MS/MS analysis has identified phosphorylation of serine residues in the C-terminal portion of GSTP1 where JNK binds, suggesting this modification regulates GSTP1-JNK interactions .

• Heterogeneity: MALDI-TOF MS analysis reveals heterogeneity in GSTP1 monomers from various sources, with secondary peaks differing from primary peaks by 194-210 atomic mass units, indicating consistent patterns of modification .

• Functional consequences: These modifications likely alter GSTP1's:

  • Binding affinity for JNK and other protein partners

  • Subcellular localization

  • Stability and turnover rate

  • Catalytic properties

To study GSTP1 post-translational modifications, researchers can use:

• Mass spectrometry approaches (MALDI-TOF, LC-MS/MS) to identify specific modifications

• Phospho-specific antibodies for detection of phosphorylated forms

• Site-directed mutagenesis of modification sites to create phospho-null or phosphomimetic variants

• 2D gel electrophoresis to separate modified forms based on charge and mass differences

What are the optimal methods for isolating and characterizing monomeric versus dimeric GSTP1 in mouse tissues?

Isolating and distinguishing between monomeric and dimeric GSTP1 requires specific methodological approaches:

• Isolation techniques:

  • Gel filtration chromatography under non-denaturing conditions to separate based on size

  • GSH-affinity chromatography to purify total GSTP1 (both monomers and dimers)

  • Non-reducing gel electrophoresis to maintain dimeric structures during separation

  • Ultracentrifugation for separation based on sedimentation coefficients

• Characterization methods:

  • MALDI-TOF mass spectrometry to determine molecular masses (monomers ~23.3 kDa, dimers ~46.5 kDa)

  • Western blotting under non-reducing conditions to preserve dimers

  • ELISA with specific antibodies for quantification

  • Native PAGE to separate based on native conformation

• Functional analysis:

  • Enzymatic activity assays (dimers are catalytically active, monomers generally inactive)

  • JNK binding assays (monomers appear to be the primary JNK-binding form)

  • Cross-linking studies to stabilize protein-protein interactions

  • Native mass spectrometry to analyze complexes in their native state

Research has shown that both monomeric and dimeric GSTP1 can be isolated from mouse tissues, with the monomeric form being particularly important for JNK regulation .

How can researchers effectively study GSTP1 phosphorylation dynamics in mouse models?

Studying GSTP1 phosphorylation dynamics requires multiple complementary approaches:

• Identification of phosphorylation sites:

  • LC-MS/MS analysis of tryptic or chymotryptic digests of purified GSTP1

  • Phosphopeptide enrichment using TiO2 or IMAC prior to MS analysis

  • Phospho-specific antibodies for known phosphorylation sites

  • Edman degradation combined with 32P labeling for precise site mapping

• Temporal dynamics analysis:

  • Time-course studies following stimulation or stress induction

  • Pulse-chase labeling with 32P to track phosphorylation turnover

  • Pharmacological manipulation of kinases and phosphatases

  • In vitro kinase assays to identify responsible kinases

• Functional consequences assessment:

  • Site-directed mutagenesis of phosphorylation sites (Ser→Ala or Ser→Asp)

  • JNK binding assays with phosphorylated versus non-phosphorylated GSTP1

  • Subcellular localization studies using fractionation or imaging approaches

  • Correlation of phosphorylation status with cellular responses

LC-MS/MS analysis has successfully identified phosphorylation of serine residues in the C-terminal portion of GSTP1 where JNK binds, providing important insights into the regulation of GSTP1-JNK interactions in mouse cells .

What experimental approaches are best for investigating tissue-specific roles of GSTP1 in mouse models?

To investigate tissue-specific roles of GSTP1, researchers can employ these experimental approaches:

• Genetic manipulation strategies:

  • Tissue-specific conditional knockout models using Cre-loxP systems

  • Tissue-specific overexpression using appropriate promoters

  • Knock-in models with tissue-specific reporters fused to GSTP1

  • CRISPR/Cas9-mediated tissue-specific gene editing

• Ex vivo tissue analysis:

  • Primary cell cultures from specific tissues of wild-type and knockout mice

  • Organoid cultures to preserve tissue architecture and cellular interactions

  • Precision-cut tissue slices for maintaining native tissue organization

  • Laser capture microdissection for analysis of specific cell populations

• In vivo functional assessment:

  • Tissue-specific responses to oxidative stress inducers

  • Analysis of tissue-specific proliferation rates and cell turnover

  • Assessment of tissue-specific JNK and ERK activation patterns

  • Pharmacological challenges with tissue-targeted GSTP1 inhibitors

• Molecular profiling:

  • Tissue-specific transcriptomics to identify GSTP1-dependent gene expression

  • Proteomics to identify tissue-specific GSTP1 interaction partners

  • Metabolomics to assess tissue-specific impact on detoxification processes

  • Phosphoproteomics to map tissue-specific signaling consequences

How should researchers design experiments to assess GSTP1's role in stress responses?

Effective experimental design for assessing GSTP1's role in stress responses should include:

• Model selection:

  • GSTP1 knockout mice compared with wild-type littermates

  • Cell-specific knockouts for tissue-focused studies

  • Knockout mice reconstituted with wild-type or mutant GSTP1 for rescue experiments

  • Knockin models with tagged GSTP1 for tracking stress-induced changes

• Stress induction protocols:

  • Oxidative stress inducers (H2O2, paraquat, menadione)

  • Xenobiotic exposures relevant to GSTP1 substrates

  • Heat shock or hypoxia for physiological stress

  • Inflammatory stress using LPS or cytokine administration

  • Time-course analyses to distinguish immediate vs. adaptive responses

• Readout parameters:

  • JNK phosphorylation and activation of downstream targets

  • ERK1/ERK2 activation status

  • Cell survival/apoptosis quantification

  • Oxidative damage markers (lipid peroxidation, protein carbonylation)

  • Glutathione levels and redox status

  • GSTP1-JNK complex dissociation kinetics

• Control considerations:

  • Dose-response relationships for stress inducers

  • Tissue-specific responses accounting for differential GSTP1 expression

  • Temporal dynamics capturing both acute and adaptive responses

  • Pharmacological intervention with JNK or ERK inhibitors for pathway validation

What controls and validation steps are critical when studying GSTP1 inhibitors in mouse models?

When studying GSTP1 inhibitors in mouse models, these controls and validation steps are essential:

• Compound validation:

  • In vitro biochemical assays to confirm direct GSTP1 inhibition

  • Determination of IC50 values and selectivity profiles against other GST isoforms

  • Pharmacokinetic analysis to establish appropriate dosing regimens

  • Verification of target engagement in vivo using activity-based probes

• Critical controls:

  • GSTP1 knockout mice as negative controls for inhibitor specificity

  • Dose-response studies to establish pharmacological relationships

  • Vehicle-only controls matched for administration route and schedule

  • Wild-type mice of matched genetic background, age, and sex

• Biological validation:

  • Confirmation of expected cellular responses (e.g., lymphocyte production, bone marrow progenitor proliferation)

  • JNK activation status assessment following inhibitor treatment

  • ERK pathway analysis to detect expected signaling changes

  • Comparison of inhibitor effects with genetic knockout phenotypes

• Potential confounding factors:

  • Strain-specific differences in GSTP1 expression or function

  • Age-dependent changes in GSTP1 levels or post-translational modifications

  • Sex-specific responses to GSTP1 inhibition

  • Environmental factors that might influence stress response pathways

The peptidomimetic inhibitor TLK199 (γ-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester) has been validated as a GSTP1 inhibitor that stimulates lymphocyte production and bone marrow progenitor proliferation in wild-type but not in GSTP1 knockout mice, confirming its specificity .

How can researchers effectively analyze contradictory data in GSTP1 mouse studies?

When faced with contradictory data in GSTP1 research, researchers should:

• Evaluate methodological differences:

  • Mouse strain variations (background genotype effects)

  • Age, sex, and environmental conditions of experimental animals

  • GSTP1 knockout strategy (global vs. conditional, targeting approach)

  • Analytical techniques (sensitivity, specificity, dynamic range)

  • Timing of measurements relative to interventions

• Consider biological complexity:

  • Compensatory mechanisms in chronic vs. acute GSTP1 deficiency

  • Context-dependent functions of GSTP1 (stress vs. basal conditions)

  • Tissue-specific expression patterns and functions

  • Post-translational modification differences between experimental systems

  • Monomeric vs. dimeric GSTP1 ratio variations

• Perform reconciliation experiments:

  • Side-by-side comparison of contradictory protocols

  • Molecular characterization of GSTP1 forms present in different systems

  • Time-course studies to capture dynamic transitions

  • Combined approaches that simultaneously measure multiple parameters

  • Genetic rescue experiments with wild-type or mutant GSTP1

• Statistical and reporting considerations:

  • Power analysis to ensure adequate sample sizes

  • Appropriate statistical methods for the specific data distributions

  • Transparent reporting of all experimental conditions

  • Publication of negative and contradictory results

  • Meta-analysis of published data when sufficient studies exist