Recombinant Human Fructose-2,6-bisphosphatase TIGAR protein (TIGAR) (Active)

What is the basic structure and function of TIGAR protein?

TIGAR (TP53-induced glycolysis and apoptosis regulator) is a 270-amino acid protein that functions primarily as a fructose-2,6-bisphosphatase. The protein contains critical catalytic domains including the RHG motif (10RHG12) and PFK motif (193PFK210) that are essential for its enzymatic activity. TIGAR negatively regulates glycolysis by lowering intracellular levels of fructose-2,6-bisphosphate, which redirects glucose metabolism toward the pentose phosphate pathway (PPP), increasing NADPH production and contributing to cellular antioxidant defenses .

How does the subcellular localization of TIGAR affect its function?

TIGAR exhibits dynamic subcellular localization across the cytoplasm, nucleus, and mitochondria, which directly influences its functional outcomes. The protein's C-terminal region (residues 258-261) is particularly important for mitochondrial localization, as TIGAR∆258–261 mutants maintain fructose-2,6-bisphosphatase activity but cannot localize to mitochondria or interact with hexokinase to protect mitochondrial function. This compartmentalization allows TIGAR to differently regulate metabolism and ROS levels depending on its location, making its trafficking an important consideration in experimental design .

What are the known structural domains of TIGAR that affect its enzymatic activity?

TIGAR's enzymatic activity depends on several critical structural domains. Deletion studies have identified that the 10RHG12 (TIGAR-∆RHG) or 193PFK210 (TIGAR-∆PFK) regions are essential for function, as mutants lacking these domains cannot reduce cell apoptosis. Additionally, the triple mutation of H11A, E102A, and H198A in TIGAR protein produces mutants that lose the ability to hydrolyze fructose-2,6-bisphosphate. Another key residue is tyrosine-92, as Y92A mutants formed by tyrosine nitration interfere with TIGAR's ability to increase PPP-dependent NADPH production .

What is the substrate specificity of TIGAR, and how does it compare to other phosphatases?

While TIGAR was initially characterized as a fructose-2,6-bisphosphatase, research has revealed that it has broader substrate specificity, with activity against 2,3-bisphosphoglycerate (2,3-BPG), 2-phosphoglycerate, phosphoglycolate, and phosphoenolpyruvate. Notably, TIGAR's enzymatic activity is approximately 400-fold higher for 2,3-BPG than for fructose-2,6-bisphosphate (Fru-2,6-P2), suggesting that 2,3-BPG might be a more physiologically relevant substrate. When designing experiments to assess TIGAR function, researchers should consider this substrate preference hierarchy rather than focusing exclusively on fructose-2,6-bisphosphate metabolism .

How can researchers accurately measure TIGAR's enzymatic activity in different experimental contexts?

To accurately measure TIGAR's enzymatic activity, researchers should employ multiple complementary approaches. For in vitro assays, purified recombinant TIGAR protein (>96% purity by SDS-PAGE and HPLC) can be used to measure phosphatase activity against different substrates using colorimetric or fluorometric detection of released phosphate. The biological activity can be assessed by its ability to protect cells (e.g., U2OS cells) from apoptosis induced by hydrogen peroxide, with effective concentration ranges typically between 0.1-5.0 μg/ml after pretreatment for 4 hours. When designing these experiments, controls should include enzymatically inactive mutants (H11A/E102A/H198A triple mutant) to distinguish between enzymatic and non-enzymatic functions .

What post-translational modifications affect TIGAR activity and how should these be considered in experimental design?

Post-translational modifications significantly impact TIGAR function. Tyrosine nitration at position 92 (Y92) interferes with TIGAR's ability to enhance PPP-dependent NADPH production. Researchers investigating TIGAR function should consider monitoring these modifications using techniques such as western blotting with modification-specific antibodies or mass spectrometry. Additionally, when working with recombinant TIGAR, researchers should verify whether their expression system reproduces relevant post-translational modifications present in the biological context being studied. The use of site-directed mutagenesis to create modification-mimicking or modification-resistant variants can help elucidate the functional significance of specific modifications in experimental settings .

How does TIGAR regulate the balance between glycolysis and the pentose phosphate pathway?

TIGAR regulates metabolic flux by inhibiting glycolysis and promoting the pentose phosphate pathway (PPP). By dephosphorylating fructose-2,6-bisphosphate, TIGAR reduces the activation of phosphofructokinase-1 (PFK1) and relieves the inhibition of fructose-1,6-bisphosphatase, leading to decreased glycolytic flux. This results in increased levels of fructose-6-phosphate that can be isomerized to glucose-6-phosphate and directed into the PPP. Additionally, TIGAR has been found to increase the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP. The resulting enhanced PPP flux increases production of NADPH and ribose-5-phosphate, supporting cellular redox balance and nucleotide synthesis. When studying this metabolic switch, researchers should measure multiple parameters including Fru-2,6-P2 levels, PPP flux (using isotope tracing), NADPH/NADP+ ratios, and G6PD activity to comprehensively characterize TIGAR's metabolic effects .

What methodologies are most effective for studying TIGAR's impact on cellular redox state?

To effectively study TIGAR's impact on cellular redox state, researchers should employ multiple complementary approaches. Direct measurement of intracellular reactive oxygen species (ROS) can be performed using fluorescent probes such as DCFDA or MitoSOX (for mitochondrial ROS). Cellular antioxidant capacity should be assessed by measuring reduced/oxidized glutathione (GSH/GSSG) ratios, NADPH/NADP+ ratios, and the activities of antioxidant enzymes like glutathione peroxidase and superoxide dismutase. Additionally, isotope tracing using 13C-labeled glucose can quantify flux through the PPP, which generates NADPH for antioxidant defense. When manipulating TIGAR expression (knockdown or overexpression), researchers should verify effects on redox state under both basal and stress conditions (e.g., H2O2 exposure) to fully capture its physiological role in redox regulation .

How do researchers distinguish between TIGAR's effects on metabolism versus its direct antioxidant functions?

Distinguishing between TIGAR's metabolic and direct antioxidant functions requires careful experimental design. Researchers should use specific TIGAR mutants that selectively disrupt either its phosphatase activity (H11A/E102A/H198A triple mutant) or its ability to localize to different cellular compartments (TIGAR∆258–261 for mitochondrial localization). Comparing these mutants' effects on ROS levels, PPP flux, and cell survival during oxidative stress can help separate direct from metabolism-mediated antioxidant functions. Metabolic flux analysis using isotope-labeled substrates (e.g., 13C-glucose) can determine whether TIGAR's effects on ROS are mediated through altered metabolic pathways. Additionally, time-course experiments can help establish whether changes in redox state precede or follow metabolic alterations, providing insights into the causal relationship between these processes .

How does TIGAR regulate NF-κB signaling independently of its phosphatase activity?

TIGAR regulates NF-κB signaling through a non-enzymatic mechanism involving direct protein-protein interactions. Research has shown that TIGAR potently inhibits NF-κB-dependent gene expression by suppressing IKKβ phosphorylation and activation. This occurs through a direct binding competition between NEMO (NF-κB essential modulator) and TIGAR for association with the linear ubiquitination assembly complex (LUBAC). When TIGAR binds to LUBAC, it prevents the linear ubiquitination of NEMO, which is required for activation of IKKβ and downstream NF-κB signaling. Importantly, phosphatase activity-deficient TIGAR mutants are equally effective as wild-type TIGAR in inhibiting this pathway, confirming that this function is independent of its enzymatic activity. Researchers studying TIGAR's role in inflammation should assess NEMO linear ubiquitination, IKKβ phosphorylation, and NF-κB target gene expression when manipulating TIGAR levels .

What experimental approaches can distinguish between TIGAR's enzymatic and non-enzymatic functions?

To distinguish between TIGAR's enzymatic and non-enzymatic functions, researchers should employ a combination of mutational analysis and pathway-specific readouts. The triple mutant (H11A, E102A, and H198A) lacking phosphatase activity is a critical tool, as it allows researchers to determine which cellular effects persist despite the loss of enzymatic function. For metabolic functions, researchers should compare how wild-type and phosphatase-dead TIGAR affect glycolytic flux, pentose phosphate pathway activity, and NADPH production. For signaling functions, measuring protein-protein interactions (through co-immunoprecipitation or proximity ligation assays) and downstream pathway activation (like NF-κB target gene expression or NEMO linear ubiquitination) with both wild-type and mutant TIGAR can reveal enzymatic-independent functions. Time-course experiments can also help distinguish primary (direct) from secondary (metabolic-dependent) effects of TIGAR expression .

How does TIGAR interact with mitochondrial proteins to regulate cellular energy metabolism?

TIGAR interacts with several mitochondrial proteins to regulate energy metabolism and mitochondrial function. One key interaction is with hexokinase 2 (HK2), which requires the C-terminal region of TIGAR (residues 258-261) for proper localization to mitochondria. This interaction protects against mitochondrial ROS production during hypoxia through a mechanism independent of TIGAR's fructose-bisphosphatase activity. TIGAR also interacts with ATP5A1, a component of the mitochondrial ATP synthase complex, potentially influencing oxidative phosphorylation. In studying these interactions, researchers should perform subcellular fractionation to confirm mitochondrial localization of TIGAR, use co-immunoprecipitation to verify protein-protein interactions, and assess functional outcomes like mitochondrial membrane potential, oxygen consumption rate, and ATP production. Comparing wild-type TIGAR with the TIGAR∆258–261 mutant can specifically isolate the effects of mitochondrial interactions .

What is TIGAR's role in neuroprotection during cerebral ischemia-reperfusion injury?

TIGAR plays a significant neuroprotective role during cerebral ischemia-reperfusion injury through multiple mechanisms. Studies have shown that TIGAR can increase the survival rate of stroke animals, improve motor function, and promote cognitive recovery. The protection appears to work through enhancing pentose phosphate pathway (PPP) flux, which generates NADPH for antioxidant defense and preserves mitochondrial function during the oxidative stress that occurs during reperfusion. By lowering ROS levels, TIGAR helps prevent oxidative damage to neurons, particularly in the context of the high metabolic demands and vulnerability of brain tissue. Researchers investigating TIGAR in stroke models should assess both acute outcomes (infarct volume, neurological deficits) and long-term recovery (cognitive function, motor skills), while simultaneously measuring markers of oxidative stress, mitochondrial function, and PPP activity to establish mechanistic links .

How does TIGAR function differently in normal versus cancer cells, and what are the implications for cancer research?

TIGAR functions differently in normal versus cancer cells due to contextual factors such as p53 status, metabolic requirements, and baseline ROS levels. In normal cells, TIGAR is typically p53-inducible and serves protective functions against oxidative and metabolic stress. In cancer cells, TIGAR can be upregulated independent of p53 status and may promote tumor survival by enhancing antioxidant capacity and nucleotide synthesis through the PPP. In glioblastomas, TIGAR protects cancer cells from hypoxia- and ROS-induced cell death by inhibiting glycolysis and activating mitochondrial energy metabolism in a TKTL1-dependent manner. TIGAR also promotes DNA repair in cancer cells through activating PPP flux in a CDK5-ATM-dependent signaling pathway. Researchers should examine TIGAR's expression patterns across different cancer types and correlate these with p53 status, glycolytic phenotype, and patient outcomes. Contrasting TIGAR's metabolic effects in matched normal and cancer cells can provide insights into cancer-specific vulnerabilities that might be therapeutically targeted .

What methodological approaches are most effective for studying TIGAR's role in inflammatory conditions?

To effectively study TIGAR's role in inflammatory conditions, researchers should implement a multi-level approach. At the molecular level, investigation should focus on TIGAR's interaction with the LUBAC complex and subsequent effects on NEMO linear ubiquitination and IKKβ activation, using co-immunoprecipitation, ubiquitination assays, and kinase activity measurements. At the cellular level, researchers should assess how TIGAR affects inflammatory cytokine production, NF-κB nuclear translocation, and expression of NF-κB target genes in relevant cell types (e.g., macrophages, adipocytes). In vivo models should include both loss-of-function (TIGAR knockout) and gain-of-function (tissue-specific TIGAR overexpression) approaches, as demonstrated in studies showing enhanced adipose tissue NF-κB signaling in TIGAR knockout mice versus suppressed signaling in adipocyte-specific TIGAR overexpression models. Time-course analyses during inflammatory challenges (e.g., LPS stimulation) can reveal the kinetics of TIGAR's anti-inflammatory effects .

What are the critical considerations when designing TIGAR overexpression systems for in vivo studies?

When designing TIGAR overexpression systems for in vivo studies, researchers must carefully consider several factors. First, the promoter choice is critical—constitutive promoters (like CAG used in the Hprt locus targeting vector) provide consistent expression, while inducible systems offer temporal control. Second, tissue specificity should be achieved using tissue-specific promoters or Cre-loxP systems (as seen in the lox-stop-lox TIGAR cDNA design) to target specific cell populations. Third, expression levels should be physiologically relevant, as extreme overexpression may cause artifacts. Fourth, researchers should include proper controls, including phosphatase-dead mutants and localization-deficient variants, to distinguish between different TIGAR functions. Finally, validation of the overexpression model should include confirmation of both TIGAR mRNA and protein levels, subcellular localization, and functional outcomes (PPP activity, ROS levels, etc.) in the targeted tissues .

What controls and validation steps are essential when working with recombinant TIGAR protein in vitro?

When working with recombinant TIGAR protein in vitro, several controls and validation steps are essential. First, protein purity should be verified using multiple methods (e.g., SDS-PAGE and HPLC), with >96% purity being the standard for reliable results. Second, endotoxin levels should be measured using the LAL method and kept below 1.0 EU/μg to prevent confounding inflammatory effects in biological assays. Third, the biological activity should be validated using functional assays, such as protection of U2OS cells from hydrogen peroxide-induced apoptosis at concentrations of 0.1-5.0 μg/ml. Fourth, protein stability should be monitored under experimental conditions, with considerations for aliquoting, storage temperature (-20°C/-80°C), and avoidance of repeated freeze-thaw cycles. Fifth, appropriate buffer conditions are critical, with recombinant TIGAR typically reconstituted from lyophilized powder in deionized sterile water to 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage. Finally, including enzymatically inactive TIGAR mutants as controls allows researchers to distinguish between enzymatic and structural effects .

How can researchers best address the contradictory data regarding TIGAR's effects on glycolysis versus oxidative phosphorylation?

Addressing contradictory data regarding TIGAR's effects on metabolism requires a comprehensive experimental approach that accounts for context-dependent factors. First, researchers should perform simultaneous measurements of multiple metabolic parameters, including glycolytic flux (extracellular acidification rate), oxidative phosphorylation (oxygen consumption rate), PPP activity (using 13C-glucose tracing), and key metabolite levels (Fru-2,6-P2, 2,3-BPG). Second, the relative expression and activity of multiple enzymes affected by TIGAR's substrates should be assessed, including PFK1, FBP, and phosphoglycerate mutase (PGM), as their relative contributions determine whether TIGAR promotes or inhibits glycolysis. Third, cell type-specific effects should be considered, as TIGAR may have different functions in different tissues (e.g., neurons versus cancer cells). Fourth, metabolic state (normoxia versus hypoxia) dramatically influences TIGAR's effects and should be carefully controlled. Finally, subcellular localization of TIGAR should be monitored, as cytosolic versus mitochondrial TIGAR may have opposing effects on metabolic pathways. By systematically addressing these variables, researchers can resolve apparent contradictions in the literature .

How is TIGAR expression regulated at the transcriptional and post-transcriptional levels?

TIGAR expression is regulated through complex transcriptional and post-transcriptional mechanisms. At the transcriptional level, p53 is a primary regulator, with TIGAR being identified as a p53-inducible gene. The TIGAR promoter contains binding sites for p53, allowing direct regulation in response to cellular stress. At the post-transcriptional level, several microRNAs regulate TIGAR expression. MiR-885-5p can directly bind to two complementary sites in the TIGAR 5'-UTR region at positions 1370 and 1310 bp from the transcription start site. This interaction increases the affinity of the TIGAR chromatin conformation and enriches RNA polymerase II in the transcriptionally active region of the promoter, ultimately regulating TIGAR expression. Additionally, miR-101 functionally interacts with the TIGAR 3'-UTR, downregulating both mRNA and protein expression of TIGAR. The precursor of miR-885 (pre-miR-885) also affects TIGAR expression, with Dicer indirectly regulating TIGAR by processing pre-miR-885 into mature miR-885 .

What experimental approaches can effectively track TIGAR's dynamic localization during cellular stress responses?

To effectively track TIGAR's dynamic localization during cellular stress responses, researchers should employ multiple complementary imaging and biochemical approaches. Live-cell imaging using fluorescently tagged TIGAR (e.g., GFP-TIGAR fusion proteins) allows real-time tracking of localization changes, while super-resolution microscopy provides detailed insights into specific subcellular compartments. These approaches should be validated with subcellular fractionation followed by western blotting to quantitatively assess TIGAR distribution across cytoplasmic, nuclear, and mitochondrial fractions before and during stress. Proximity ligation assays can identify specific protein interactions in different compartments (e.g., with hexokinase 2 at mitochondria). When designing these experiments, researchers should include localization-deficient mutants (e.g., TIGAR∆258–261 for mitochondrial localization) as controls and expose cells to relevant stressors (oxidative stress, hypoxia, nutrient deprivation) to capture the full range of dynamic responses. Time-course experiments with multiple time points are essential to fully characterize the kinetics of TIGAR translocation during stress responses .