GOT1 Mouse

Basic Q: What is the primary enzymatic function of GOT1 in murine models, and how is it linked to cellular metabolism?

GOT1 (glutamate oxaloacetate transaminase 1) catalyzes the reversible transfer of amino groups between glutamate and aspartate, a critical reaction in the malate-aspartate shuttle (MAS). This shuttle transfers reducing equivalents (NADH) from the cytosol to mitochondria, enabling metabolic coordination between glycolysis and the tricarboxylic acid (TCA) cycle . In murine models, GOT1 is homodimeric, with cytoplasmic localization, and plays dual roles in amino acid metabolism and redox balance .

Key Methodological Note:
To assess GOT1 activity, use aminooxyacetate (AOA) inhibitors or genetic knockout models. For example, Xu et al. employed T cell-specific GOT1 knockout mice to demonstrate its role in glutamate synthesis from glucose-derived α-ketoglutarate .

Advanced Q: How do tissue-specific GOT1 knockout models reveal its functional diversity across organs?

Tissue-specific deletions in murine models have uncovered context-dependent roles:

• Retina: Rod photoreceptor-specific Got1 knockout causes retinal degeneration, linked to aspartate accumulation and disrupted MAS/TCA cycle gene expression .

• Immune Cells:

  • Macrophages: Got1 deletion reduces ROS production but does not impair pathogen resistance, suggesting compensatory mechanisms .

  • CD8+ T Cells: Got1 KO impairs effector T cell proliferation under serine-free conditions but promotes memory T cell generation .

Experimental Design Insight:
Use Cre-Lox recombination to generate tissue-specific knockouts. For example, photoreceptor-specific deletion required rod-specific Cre lines (e.g., Pcp2-Cre) , while T cell studies used CD4-Cre or Lck-Cre .

Basic Q: What are the key metabolic pathways regulated by GOT1 in murine models?

GOT1 intersects with three core pathways:

• Malate-Aspartate Shuttle (MAS): Facilitates NADH transfer to mitochondria, sustaining oxidative phosphorylation .

• Tricarboxylic Acid (TCA) Cycle: Regulates α-ketoglutarate availability, influencing glutamate synthesis and redox balance .

• Serine Biosynthesis: Supports de novo serine production via 3-phosphoglycerate, critical for nucleotide synthesis and T cell proliferation .

Metabolomic Validation:
Use [U-13C]glucose tracing to map carbon flux. For example, Xu et al. demonstrated reduced glucose-derived glutamate in Got1-deficient CD8+ T cells, confirming MAS dependency .

Advanced Q: Why do studies report conflicting roles of GOT1 in immune and retinal tissues?

Contradictory Findings:

Key Discrepancy: Tissue-specific compensatory mechanisms. For example, macrophages may rely on alternative ROS sources (e.g., NADPH oxidase) when GOT1 is absent , whereas photoreceptors lack such redundancy .

Basic Q: How is GOT1 activity measured in murine models?

Experimental Approaches:

• Enzymatic Assays:

  • Measure transamination activity using glutamate/aspartate substrates and NADH/NAD+ coupling.

• Metabolic Tracing:

  • Track carbon flux via [U-13C]glucose or [U-13C]glutamine to quantify MAS/TCA contributions .

• Genetic Models:

  • Use GOT1 KO mice to assess phenotypic outcomes (e.g., retinal degeneration in photoreceptor-specific models) .

Validation Tip: Confirm KO efficacy via Western blot (e.g., GOT1 antibody) and qPCR (gene expression) .

Advanced Q: How does GOT1 modulate redox balance in murine immune cells?

Mechanisms:

• NADH/NAD+ Ratio: GOT1 regulates cytosolic redox via MAS, influencing glycolytic flux .

• ROS Production:

  • In macrophages, Got1 deletion reduces ROS but does not impair bacterial clearance, suggesting alternative ROS sources (e.g., NADPH oxidase) .

• α-Ketoglutarate (α-KG):

  • GOT1 converts α-KG to glutamate, modulating TCA metabolism and HIF-1α stability in T cells .

Methodological Challenge:
Distinguish between direct GOT1 effects and indirect redox shifts using combined genetic (KO) and pharmacological (AOA) approaches .

Basic Q: What are the implications of GOT1 upregulation in effector CD8+ T cells?

GOT1 upregulation in effector T cells supports:

• Serine Biosynthesis: Critical for nucleotide synthesis and proliferation under serine-depleted conditions .

• Glycolytic Programming: Enhances glucose utilization and cytotoxic function via HIF-1α stabilization .

• Effector vs. Memory Fate: High GOT1 promotes effector differentiation, while low GOT1 favors memory T cell persistence .

Experimental Model:
Use serine-free media to isolate GOT1-dependent proliferation defects in Got1-deficient T cells .

Advanced Q: How can researchers resolve contradictions between GOT1’s role in retinal vs. immune cells?

Strategic Approaches:

• Tissue-Specific Studies: Compare metabolomic profiles (e.g., aspartate/NADH levels) in retina vs. macrophages .

• Compensation Analysis: Identify alternative pathways (e.g., NADPH oxidase in macrophages) using RNA-seq or proteomics .

• Temporal Dynamics: Assess age-dependent phenotypes. For example, retinal degeneration in Got1-deficient mice progresses with age , while T cell defects are acute .

Hypothetical Table:

Basic Q: What tools are available for studying GOT1 in murine models?

Experimental Tools:

• Recombinant Proteins: His-tagged GOT1 (48.6 kDa) for biochemical assays .

• Genetic Models:

  • Global KO: Got1 null mice (e.g., Got1^-/^-).

  • Tissue-Specific KO: Got1^-/^-; Pcp2-Cre (retina) , Got1^-/^-; Lck-Cre (T cells) .

• Inhibitors:

  • AOA: Blocks GOT1 activity (e.g., in T cell cultures) .

  • EGCG: Inhibits GLUD1 (control for α-KG→glutamate flux) .

Resource Note:
Recombinant GOT1 (Cat. No. Got1-65M) is available for in vitro studies .

Advanced Q: How does GOT1 influence antigen-specific T cell responses in vivo?

Key Findings:

• Effector Function: GOT1 sustains glycolysis and cytotoxicity via α-KG-dependent HIF-1α stabilization .

• Memory Formation: Low GOT1 levels promote oxidative metabolism, favoring memory T cell persistence .

• Experimental Evidence:

  • Got1^-/^- CD8+ T cells show impaired proliferation in Listeria monocytogenes infection but retain cytotoxicity .

  • Adoptive Transfer Models: Co-transfer WT and Got1^-/^- T cells to assess serine sharing .

Methodological Challenge:
Distinguish between intrinsic GOT1 effects and extrinsic metabolite sharing using parabiosis or co-culture systems .

Basic Q: What are the redox-sensitive pathways regulated by GOT1?

Key Pathways:

• Malate-Aspartate Shuttle: Regulates NADH/NAD+ ratios, influencing glycolytic flux .

• Glutathione Synthesis: Aspartate (via GOT1) is a precursor for glutathione, a critical antioxidant .

• HIF-1α Stabilization: α-KG availability modulates prolyl hydroxylation, affecting HIF-1α degradation .

Measurement Tools:

• NADH/NAD+ Assays: Quantify redox state.

• Glutathione Quantification: Use monochlorobimane (MCB) probes .

Advanced Q: How can researchers validate GOT1-dependent metabolic flux in vivo?

Validation Strategies:

• Stable Isotope Tracing:

  • [U-13C]Glucose: Track carbon flux to glutamate (MAS activity) .

  • [U-13C]Glutamine: Assess glutamate synthesis from glutamine (GLUD1 activity) .

• Flux Analysis:

  • Metabolite Profiling: LC-MS/MS to quantify aspartate, glutamate, and serine .

• Genetic Controls:

  • Compare Got1^-/^- with GLUD1^-/^- mice to isolate GOT1-specific effects .

Example Table:

Basic Q: What are the phenotypic hallmarks of GOT1 deficiency in murine models?

Tissue-Specific Phenotypes:

Diagnostic Markers:

• Retina: Elevated aspartate, NADH accumulation .

• T Cells: Reduced 3-phosphoserine, impaired glucose-derived serine .

Advanced Q: How might GOT1 modulate cancer progression in murine models?

Hypothetical Roles:

• Tumor Microenvironment:

  • Regulate T cell metabolism (effector vs. memory bias) .

• Cancer Cell Metabolism:

  • Sustain glycolysis via MAS, promoting tumor growth.

• Experimental Models:

  • Study Got1-deficient mice in syngeneic tumor models (e.g., MC38).

  • Assess tumor-infiltrating T cell function and proliferation.

Unanswered Questions:

• Does GOT1 inhibition enhance immunotherapy efficacy?

• How does GOT1 interact with other metabolic regulators (e.g., PHGDH)?

Basic Q: What are the technical considerations for generating GOT1 knockout mice?

Key Steps:

• Targeting Vector Design:

  • Use homology-directed repair to excise exon(s) critical for GOT1 activity.

• Tissue-Specific Deletion:

  • Pair Got1^-/fl mice with Cre drivers (e.g., Pcp2-Cre, Lck-Cre) .

• Validation:

  • Genotyping: PCR for loxP sites.

  • Protein Confirmation: Western blot for GOT1 .

Pitfall: Global KO may cause embryonic lethality; use conditional models.