GOT2 Human

What is the biochemical function of GOT2 in human cellular metabolism?

GOT2 (Glutamic-Oxaloacetic Transaminase 2) is a mitochondrial enzyme that catalyzes the reversible transamination between aspartate and α-ketoglutarate to form oxaloacetate and glutamate. This reaction is a critical component of the malate-aspartate shuttle, which facilitates the transfer of reducing equivalents between cytosolic and mitochondrial compartments to maintain cellular redox balance .

The enzyme serves several key metabolic functions:

• Facilitates electron transfer between separate cytosolic and mitochondrial NADH pools

• Contributes to amino acid metabolism, particularly aspartate biosynthesis

• Supports glutamine anaplerosis into the TCA cycle

• Helps maintain NAD+/NADH ratios to support continued glycolysis and mitochondrial respiration

In normal physiology, GOT2 primarily functions as a shuttle component, but in certain pathological conditions like cancer, its role can be reprogrammed to support alternative metabolic pathways .

How can researchers accurately measure GOT2 expression levels in human biological samples?

Several validated methodologies are available for quantifying GOT2 in research settings:

For ELISA-based detection, sandwich immunoassay techniques using polyclonal antibodies specific for human GOT2 offer high specificity and sensitivity, with detection limits as low as 1.563 ng/mL . Sample preparation protocols typically involve standard centrifugation steps for fluid samples or lysis buffers for cellular material.

What is the role of GOT2 in maintaining cellular redox balance under normal physiological conditions?

GOT2 plays a fundamental role in cellular redox homeostasis by facilitating the malate-aspartate shuttle (MAS). This biochemical system transfers reducing equivalents generated during glycolysis from cytosolic NADH to the mitochondrial electron transport chain .

The process involves:

• Cytosolic malate dehydrogenase converts oxaloacetate to malate while oxidizing NADH to NAD+

• Malate enters mitochondria via specific transporters

• Mitochondrial malate dehydrogenase converts malate to oxaloacetate, reducing NAD+ to NADH

• GOT2 converts oxaloacetate to aspartate using glutamate as an amino group donor

• Aspartate is transported out of mitochondria via the aspartate-glutamate carrier

• Cytosolic GOT1 converts aspartate back to oxaloacetate

This cycle effectively transfers reducing equivalents between compartments, maintaining appropriate NAD+/NADH ratios in both cytosolic and mitochondrial compartments, which is essential for continued glycolysis and efficient ATP production .

How does GOT2 contribute to metabolic rewiring in pancreatic ductal adenocarcinoma?

In pancreatic ductal adenocarcinoma (PDA), GOT2 becomes integral to KRAS-driven metabolic reprogramming. Research using LC-MS/MS metabolomics and genetic manipulation has revealed that mutant KRAS redirects glutamine metabolism through a non-canonical pathway .

In this rewired pathway:

• GOT2 drives the conversion of glutamine-derived metabolites

• Metabolites are diverted from the normal MAS function

• Flux is increased through malic enzyme 1 (ME1)

• This redirection produces NADPH, which is critical for managing oxidative stress in PDA cells

Experimental evidence demonstrates that GOT2 knockdown in PDA cells in vitro leads to:

• Decreased production of aspartate and α-ketoglutarate

• Disruption of TCA cycle intermediates

• Stalled glycolysis

• NADH accumulation (reductive stress)

• Impaired cellular proliferation

These effects can be rescued by supplementation with aspartate or α-ketoglutarate, confirming the role of these GOT2 products in sustaining cancer cell growth .

What experimental approaches have revealed discrepancies between in vitro and in vivo findings regarding GOT2 inhibition in cancer?

Researchers have uncovered remarkable differences in the effects of GOT2 inhibition between controlled laboratory conditions and living systems. This paradox highlights the challenge of translating in vitro findings to clinical applications.

The resolution to this paradox came through experimental manipulation of the metabolic environment. Researchers discovered that:

• Cancer-associated fibroblasts (CAFs) release pyruvate into the tumor microenvironment

• Pyruvate acts as an electron acceptor that can oxidize NADH

• CAF-conditioned media rescues growth of GOT2-knockdown cells in vitro

• Pyruvate levels in mouse serum (~250 μM) are sufficient to compensate for GOT2 loss

• Blocking pyruvate import prevents this rescue effect in vitro

These findings demonstrate remarkable metabolic plasticity and emphasize how environmental context significantly impacts cancer cell metabolism .

How can GOT2 be leveraged to improve CAR-T cell function in solid tumor microenvironments?

Engineered expression of GOT2 in CAR-T cells represents a novel approach to overcome the metabolic challenges of solid tumor environments. Preclinical research has demonstrated that GOT2-overexpressing CAR-T cells (BOXR1030) show enhanced functionality compared to conventional CAR-T cells .

The mechanistic rationale includes:

• GOT2 helps maintain cellular redox balance under oxidative stress conditions

• It fuels the tricarboxylic acid (TCA) cycle via glutaminolysis

• This metabolic enhancement supports T cell fitness in hostile tumor microenvironments

Experimental characterization of BOXR1030 cells has revealed:

• Higher frequency of T stem cell memory (CD45RA+CCR7+CD27+CD95+) populations

• Increased proportion of T central memory (CD45RO+CCR7+CD27+) in CD8+ cells

• Reduced frequency of terminally differentiated CD27-CD28- T cells

• Preservation of early memory populations after repeated stimulation

• Superior performance in both standard and low glucose conditions

These findings suggest that GOT2 engineering may delay T cell exhaustion and enhance persistence, critical factors for effective solid tumor immunotherapy .

How do cancer-associated fibroblasts influence GOT2-dependent metabolism in tumors?

Cancer-associated fibroblasts (CAFs) play a pivotal role in modulating GOT2-dependent metabolism through metabolic crosstalk with tumor cells. This relationship helps explain the discrepancy between in vitro and in vivo findings on GOT2 inhibition.

Research using co-culture systems and conditioned media has revealed that:

• CAFs constitute a substantial portion of the pancreatic tumor microenvironment, confirmed by α-smooth muscle actin (αSMA) staining

• CAFs release metabolites that can rescue GOT2-inhibited cancer cells

• Human CAF-conditioned media promotes colony formation in GOT2-knockdown PDA cells in a dose-dependent manner

• CAF-derived pyruvate serves as an electron acceptor, relieving NADH reductive stress

• This rescue effect is significantly stronger from CAFs compared to tumor-educated macrophages or cancer cells themselves

Metabolic intervention studies demonstrated that blocking pyruvate import or preventing pyruvate-to-lactate reduction abrogated the rescue effect in vitro, confirming the specific role of pyruvate in this metabolic symbiosis .

What methodological approaches can researchers use to study GOT2-dependent metabolic flux in human cancer models?

To effectively investigate GOT2-mediated metabolic processes, researchers should consider these established methodological approaches:

• Metabolomics Analysis

  • Liquid chromatography-coupled tandem mass spectroscopy (LC-MS/MS) to measure changes in metabolite pools

  • Stable isotope tracing (using 13C- or 15N-labeled precursors) to track metabolic flux through GOT2-dependent pathways

  • Targeted analysis of TCA cycle intermediates and amino acids

• Redox State Assessment

  • Measurement of NAD+/NADH ratios in cellular compartments

  • Analysis of glycolytic intermediates as indicators of pathway blockade

  • Seahorse Flux Analysis to assess real-time changes in glycolytic rate

• Genetic Manipulation

  • RNAi-mediated GOT2 knockdown in cancer cell lines

  • CRISPR/Cas9-mediated knockout in vitro and in vivo

  • Conditional knockout models (e.g., LSL-Kras^G12D;Got2^f/f;Ptf1a-Cre)

  • Expression of cytosolic NADH oxidase as a redox intervention

• Microenvironmental Modeling

  • Co-culture systems with cancer-associated fibroblasts

  • Collection and analysis of conditioned media

  • 3D culture systems (spheroids) with controlled nutrient availability

  • Xenograft and autochthonous mouse models for in vivo validation

• Metabolic Rescue Experiments

  • Supplementation with aspartate, α-ketoglutarate, or pyruvate

  • Inhibition of pyruvate transport or metabolism

  • Manipulation of glucose availability to alter glycolytic dependency

These approaches collectively enable a comprehensive understanding of GOT2's role in cancer metabolism and provide opportunities for therapeutic target validation .