C12ORF5 Human, His

What is C12ORF5/TIGAR and what are its primary biological functions?

C12ORF5/TIGAR is a 270-amino acid protein encoded by the TIGAR gene (previously known as C12orf5) located on chromosome 12p13-3 in humans. It was discovered in 2005 by Kuang-Yu Jen and Vivian G. Cheung as a p53-regulated gene activated in response to ionizing radiation . The protein functions primarily as a fructose-bisphosphatase that hydrolyzes fructose-2,6-bisphosphate and fructose-1,6-bisphosphate.

TIGAR's primary biological functions include:

• Negative regulation of glycolysis by decreasing intracellular levels of fructose-2,6-bisphosphate

• Activation of the pentose phosphate pathway (PPP), leading to increased NADPH production

• Decreasing reactive oxygen species (ROS) levels and protecting cells from oxidative stress

• Providing protection against DNA damage-induced apoptosis

• Contributing to cellular survival during hypoxic conditions

What is the structural composition of recombinant Human TIGAR/C12ORF5 His-tag protein?

Recombinant Human TIGAR/C12ORF5 His-tag protein typically consists of the human TIGAR sequence (Ala2-Arg270) with a C-terminal 6-His tag . The protein has the following structural characteristics:

• Molecular mass of approximately 32.6 kDa (including the His-tag)

• Single, non-glycosylated polypeptide chain containing 294 amino acids (270 amino acids from TIGAR sequence plus 24 amino acids from the His-tag)

• Histidine phosphatase fold with a phosphate coordinated to a catalytic histidine in the active site

• Monomeric protein under physiological conditions

The recombinant protein is typically produced in E. coli expression systems and purified using chromatographic techniques to achieve >90-95% purity as determined by SDS-PAGE .

How should researchers properly store and handle recombinant TIGAR protein to maintain stability?

For optimal stability and activity of recombinant TIGAR/C12ORF5 His-tag protein, researchers should follow these methodological guidelines:

• Short-term storage (2-4 weeks): Store at 4°C in the recommended buffer solution

• Long-term storage: Store at -20°C with proper aliquoting to avoid freeze-thaw cycles

• For extended storage periods, addition of a carrier protein (0.1% HSA or BSA) is recommended to enhance stability unless working with carrier-free preparations

Typical formulation includes:

• 20mM Tris-HCl buffer (pH 8.0)

• 0.2M NaCl

• 2mM DTT

• 10% glycerol

For carrier-free versions, researchers should be aware that the absence of stabilizing BSA may affect shelf-life but is beneficial for applications where BSA might interfere with experimental outcomes .

What are the basic assays available for measuring TIGAR enzymatic activity?

Researchers can employ several methodological approaches to measure TIGAR enzymatic activity:

• Phosphatase Activity Assay:

  • Substrate: 4-Nitrophenyl Phosphate (pNPP)

  • Detection: Spectrophotometric measurement at 410 nm

  • Calculation method: Specific Activity (pmol/min/μg) = [Adjusted Absorbance × Conversion Factor] / [Incubation time × enzyme amount]

• Fructose-2,6-bisphosphate Hydrolysis Assay:

  • Direct measurement of F2,6BP degradation

  • Detection: Coupled enzymatic assays measuring phosphofructokinase activity

• Indirect Assessment via Metabolic Flux Analysis:

  • Measurement of glycolytic intermediates

  • Analysis of pentose phosphate pathway activation

  • NADPH production quantification

A standardized protocol for phosphatase activity includes:

• Incubation of TIGAR (typically 2 μg per reaction) with 1.25 mM substrate at 37°C for 3 hours

• Reaction termination with 0.2 M NaOH

• Spectrophotometric analysis and calculation of specific activity using appropriate conversion factors and controls

How does TIGAR contribute to cancer progression, and what are the methodological approaches to study this relationship?

TIGAR demonstrates complex roles in cancer biology, exhibiting both tumor-suppressive and tumor-promoting functions depending on cellular context. Research methodologies to investigate TIGAR's role in cancer include:

• Metabolic Profiling Approaches:

  • Isotope tracing using 13C-labeled glucose to track metabolic flux through glycolysis versus pentose phosphate pathway

  • Measurement of NADPH/NADP+ ratios to assess redox status

  • Quantification of ribose-5-phosphate production for nucleotide synthesis

• In Vitro Cancer Models:

  • TIGAR knockdown/overexpression studies in cancer cell lines

  • Assessment of proliferation, migration, invasion capacity

  • Evaluation of sensitivity to chemotherapeutic agents and radiation

• In Vivo Models:

  • Xenograft studies with TIGAR-modulated cancer cells

  • Conditional knockout mouse models

  • Analysis of metastatic potential and tumor microenvironment interactions

Research findings indicate that TIGAR can promote cancer metastasis through:

• Enzymatic functions that redirect glucose metabolism toward the pentose phosphate pathway

• Interactions with signaling pathways that inhibit autophagy and apoptosis

• Nuclear translocation during chemotherapy and hypoxia, promoting cell survival

• Mitochondrial translocation during hypoxia, enhancing hexokinase 2 (HK2) activity

TIGAR is highly expressed in various tumor types and represents a potential therapeutic target for cancer treatment strategies .

How can researchers accurately distinguish between the metabolic and non-metabolic functions of TIGAR in experimental designs?

Distinguishing between TIGAR's metabolic and non-metabolic functions requires sophisticated experimental designs:

• Enzymatic Activity Mutations:

  • Generation of catalytically inactive TIGAR mutants that retain protein-protein interaction capabilities

  • Compare phenotypes between wild-type and mutant TIGAR expression

• Subcellular Localization Studies:

  • Creation of localization-restricted TIGAR variants (nuclear, cytoplasmic, mitochondrial)

  • Fluorescent tagging and live cell imaging to track translocation events

  • Correlation of localization with specific functions

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation studies to identify interaction partners in different cellular compartments

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry-based interactome analysis

• Metabolic Rescue Experiments:

  • Provision of metabolic intermediates to bypass TIGAR's enzymatic functions

  • Administration of antioxidants to neutralize ROS-related effects

  • Direct NADPH supplementation to distinguish between redox and other functions

Research has revealed that TIGAR's protective function during hypoxia occurs through a mechanism independent of its fructose-bisphosphatase activity, involving interactions with HK2 . Similarly, TIGAR's interaction with ATP5A1 reduces oxidative stress through protein-protein interactions rather than enzymatic activity .

What experimental controls are essential when working with recombinant TIGAR protein in functional assays?

When designing experiments with recombinant TIGAR protein, researchers should implement these essential controls:

For phosphatase activity assays specifically, an enzyme control prepared by combining TIGAR with NaOH before substrate addition is critical to account for background signal and ensure accurate activity calculations .

How can researchers resolve contradictory findings regarding TIGAR's role in autophagy regulation?

Contradictory findings regarding TIGAR's role in autophagy regulation reflect its context-dependent functions. Methodological approaches to resolve these contradictions include:

• Cell Type-Specific Analysis:

  • Systematic evaluation across multiple cell types (primary cells, cancer cell lines, non-transformed cells)

  • Correlation with baseline metabolic state (glycolytic vs. oxidative phosphorylation-dependent)

  • Analysis of p53 status and correlation with autophagy outcomes

• Stress Condition Stratification:

  • Precise categorization of stress stimuli (nutrient deprivation, hypoxia, DNA damage)

  • Temporal analysis of TIGAR expression and autophagy markers

  • Quantification of autophagic flux rather than static autophagosome numbers

• Molecular Mechanism Dissection:

  • Evaluation of mTOR signaling pathway components

  • Assessment of AMPK activation status

  • Analysis of Beclin1 complexes and interacting partners

• In Vivo Validation:

  • Tissue-specific conditional knockout models

  • Physiological versus pathological stress conditions

  • Correlation with clinical samples and patient outcomes

Research has demonstrated that TIGAR mediates p53-induced inhibition of myocyte mitophagy through ROS reduction and subsequent inactivation of BNIP3, which can exacerbate cardiac damage . Conversely, in other contexts, TIGAR may promote autophagy to maintain cellular homeostasis, highlighting the importance of experimental context in interpreting results.

What are the optimal expression and purification protocols for generating high-quality recombinant TIGAR protein?

The production of high-quality recombinant TIGAR protein involves several methodological considerations:

• Expression System Selection:

  • E. coli BL21(DE3) is commonly used for TIGAR expression

  • Consider codon optimization for efficient expression

  • Alternative systems (insect cells, mammalian cells) may be necessary for specific applications requiring post-translational modifications

• Expression Vector Design:

  • Incorporate appropriate promoter (T7, tac)

  • Include affinity tag (6xHis) for purification

  • Consider fusion partners (GST, MBP) for enhanced solubility

  • Include precision protease cleavage sites if tag removal is required

• Expression Conditions Optimization:

  • Test multiple induction temperatures (16°C, 25°C, 37°C)

  • Vary IPTG concentrations (0.1-1.0 mM)

  • Optimize expression duration (3-24 hours)

  • Consider auto-induction media for higher yields

• Purification Protocol:

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Secondary purification: Size exclusion chromatography

  • Quality control: SDS-PAGE, Western blotting, mass spectrometry

  • Activity verification: Enzymatic assays

• Buffer Optimization:

  • Final formulation typically includes 20mM Tris-HCl (pH 8.0), 0.2M NaCl, 2mM DTT, and 10% glycerol

  • Stability testing under various storage conditions

  • Compatibility assessment with downstream applications

For optimal results, researchers should verify protein purity (>90% by SDS-PAGE) and confirm enzymatic activity through phosphatase assays before using in experimental procedures .

How can researchers effectively study TIGAR's role in the pentose phosphate pathway and redox regulation?

Studying TIGAR's impact on the pentose phosphate pathway (PPP) and redox regulation requires integrated methodological approaches:

• Metabolic Flux Analysis:

  • Use of 13C-labeled glucose to track carbon flow through glycolysis versus PPP

  • GC-MS or LC-MS/MS analysis of labeled metabolites

  • Computational modeling of flux distributions

• Redox Status Assessment:

  • Quantification of NADPH/NADP+ ratio using enzymatic assays or targeted metabolomics

  • Measurement of reduced/oxidized glutathione levels (GSH/GSSG)

  • ROS detection using fluorescent probes (CM-H2DCFDA, MitoSOX)

  • Protein oxidation assessment (protein carbonylation, thiol oxidation)

• Gene Expression and Protein Analysis:

  • Quantification of PPP enzyme expression (G6PD, 6PGD, TKT)

  • Assessment of antioxidant defense systems (glutathione peroxidase, peroxiredoxins)

  • Analysis of redox-sensitive transcription factors (Nrf2, NF-κB)

• Functional Outcomes Measurement:

  • Cell viability under oxidative stress conditions

  • DNA damage assessment (comet assay, γH2AX foci)

  • Mitochondrial function analysis (oxygen consumption rate, membrane potential)

• Integration with Disease Models:

  • Cancer cells with varying metabolic dependencies

  • Ischemia-reperfusion injury models

  • Neurodegenerative disease models sensitive to oxidative stress

Research has established that TIGAR contributes to the generation of reduced glutathione to decrease intracellular ROS, correlating with its ability to protect cells from oxidative stress-induced death . Additionally, TIGAR plays a neuroprotective role against ischemic brain damage by enhancing PPP flux and preserving mitochondrial functions .

What advanced imaging techniques can be employed to study TIGAR subcellular localization and dynamics?

Advanced imaging techniques provide crucial insights into TIGAR's subcellular localization and dynamics:

• Fluorescent Fusion Protein Approaches:

  • Generation of TIGAR-GFP/mCherry fusion constructs

  • Lentiviral transduction for stable expression

  • Live-cell confocal microscopy for dynamic tracking

  • Photoactivatable or photoconvertible tags for pulse-chase localization studies

• Super-Resolution Microscopy:

  • Stimulated emission depletion (STED) microscopy for nanoscale resolution

  • Stochastic optical reconstruction microscopy (STORM) for single-molecule localization

  • Structured illumination microscopy (SIM) for enhanced resolution of subcellular structures

• Multi-Channel Co-Localization Analysis:

  • Simultaneous imaging of TIGAR with organelle markers:

    • Mitochondria: MitoTracker, TOM20 antibodies

    • Nucleus: DAPI, Hoechst

    • ER: Calnexin, PDI

    • Lysosomes: LAMP1, LysoTracker

  • Quantitative co-localization metrics (Pearson's coefficient, Manders' overlap)

• Förster Resonance Energy Transfer (FRET):

  • Detection of protein-protein interactions in live cells

  • Measurement of conformational changes in TIGAR protein

  • Biosensor development for TIGAR activity

• Fluorescence Recovery After Photobleaching (FRAP):

  • Assessment of TIGAR mobility within cellular compartments

  • Determination of binding dynamics to subcellular structures

Research has demonstrated that TIGAR translocates to different subcellular compartments under specific conditions:

• Nuclear translocation promotes cell survival during chemotherapy and hypoxia

• Mitochondrial translocation under hypoxia enhances HK2 activity and maintains cell survival

• Interaction with ATP5A1 in mitochondria reduces oxidative stress

These dynamic localizations highlight the importance of advanced imaging techniques in understanding TIGAR's multifaceted functions.

How can researchers address variability in TIGAR phosphatase activity assays?

Variability in TIGAR phosphatase activity assays presents a common challenge. Methodological solutions include:

• Standardization of Protein Quality:

  • Implement batch-to-batch quality control testing

  • Verify protein purity by SDS-PAGE and Western blotting

  • Perform thermal shift assays to confirm proper folding

  • Use freshly thawed aliquots for each experiment

• Reaction Condition Optimization:

  • Systematically test buffer components:

    • pH range (6.5-8.5)

    • Salt concentration (50-300 mM NaCl)

    • Divalent cations (Mg2+, Mn2+)

    • Reducing agents (DTT, β-mercaptoethanol)

  • Determine optimal enzyme:substrate ratio

  • Establish temperature dependence (25°C, 30°C, 37°C)

• Substrate Considerations:

  • Use freshly prepared substrate solutions

  • Verify substrate purity by analytical methods

  • Consider competing reactions or substrate instability

  • Include substrate-only controls in every experiment

• Advanced Data Analysis:

  • Apply baseline correction algorithms

  • Calculate initial velocities from linear portions of progress curves

  • Use internal standards for assay normalization

  • Consider mathematical modeling of reaction kinetics

• Technical Replicates and Controls:

  • Perform technical triplicates for each condition

  • Include positive controls with known activity

  • Implement enzyme controls to account for background signal

  • Perform inter-day validation experiments

Following the standardized protocol described in source , which includes appropriate enzyme controls and specific activity calculations, can significantly reduce assay variability and improve reproducibility.

What are the common pitfalls when investigating TIGAR's role in cancer metabolism and how can they be overcome?

When investigating TIGAR's role in cancer metabolism, researchers encounter several methodological challenges:

• Cell Line Heterogeneity:

  • Pitfall: Varying baseline TIGAR expression and p53 status across cancer cell lines

  • Solution: Systematically characterize multiple cell lines before selection; create isogenic cell line panels; document p53 status

• Metabolic Adaptation:

  • Pitfall: Compensatory metabolic pathways activated upon TIGAR modulation

  • Solution: Employ acute (inducible) rather than chronic TIGAR modulation; perform time-course studies; analyze multiple metabolic pathways simultaneously

• In Vitro vs. In Vivo Discrepancies:

  • Pitfall: Cell culture conditions poorly recapitulate tumor microenvironment

  • Solution: Use 3D culture systems; implement co-culture with stromal cells; validate findings in patient-derived xenografts

• Technical Limitations in Metabolite Analysis:

  • Pitfall: Rapid turnover and instability of key metabolites

  • Solution: Optimize rapid quenching techniques; employ stable isotope tracing; integrate computational modeling with experimental data

• Contextual Dependency:

  • Pitfall: TIGAR functions vary with nutrient availability and oxygen tension

  • Solution: Precisely control and document culture conditions; systematically test nutrient limitation and hypoxia; correlate with in vivo tumor regions

Research has shown that TIGAR protects glioma cells from hypoxia and ROS-induced cell death by inhibiting glycolysis and activating mitochondrial energy metabolism in a TKTL1-dependent manner . Additionally, TIGAR plays a role in cancer cell survival by promoting DNA repair through activating PPP flux in a CDK5-ATM-dependent signaling pathway during hypoxia and/or genome stress-induced DNA damage responses .

How can researchers effectively distinguish between specific TIGAR effects and general metabolic perturbations?

Distinguishing specific TIGAR effects from general metabolic perturbations requires methodological rigor:

• Genetic Approach Considerations:

  • Complementary Techniques: Employ both knockdown (siRNA, shRNA) and knockout (CRISPR-Cas9) approaches

  • Rescue Experiments: Re-express TIGAR wild-type or mutant variants to confirm specificity

  • Dose-Response Analysis: Establish graded expression levels to identify threshold effects

• Metabolic Intervention Design:

  • Targeted Metabolite Supplementation: Provide specific metabolites (ribose-5-phosphate, NADPH) to bypass TIGAR effects

  • Parallel Pathway Modulation: Simultaneously manipulate alternative metabolic enzymes (G6PD, PKM2)

  • Metabolic Inhibitors: Use specific inhibitors of glycolysis or PPP to dissect pathway contributions

• Temporal Analysis:

  • Early vs. Late Responses: Distinguish immediate TIGAR effects from adaptive responses

  • Pulse-Chase Experiments: Track metabolic flux changes over time using labeled precursors

  • Inducible Systems: Employ temporal control of TIGAR expression or activity

• Multi-Omics Integration:

  • Correlative Analysis: Integrate transcriptomics, proteomics, and metabolomics data

  • Network Analysis: Apply pathway enrichment and network modeling to identify specific nodes

  • Single-Cell Technologies: Assess heterogeneity of responses within cell populations

• Direct Target Validation:

  • In Vitro Enzyme Assays: Confirm direct enzymatic activities with purified components

  • Target Engagement Studies: Assess physical interactions in cellular contexts

  • Structure-Function Analysis: Utilize domain-specific mutations to dissect activities

Research indicates that TIGAR has both enzymatic and non-enzymatic functions, including interactions with mitochondrial proteins that occur independently of its fructose-bisphosphatase activity . Careful experimental design is essential to distinguish these diverse mechanistic effects.

What are the most promising research directions for TIGAR in disease treatment and prevention?

Current evidence points to several promising research directions for TIGAR in disease contexts:

• Cancer Therapeutics Development:

  • Targeting TIGAR in metabolically flexible cancers that rely on PPP activation

  • Combination approaches with standard chemotherapies to prevent resistance

  • Synthetic lethality strategies in p53-mutated cancers

  • Development of specific TIGAR inhibitors as potential anticancer agents

• Cardiovascular Disease Interventions:

  • Modulation of TIGAR to protect against ischemia-reperfusion injury

  • Targeted approaches to prevent excessive TIGAR-mediated inhibition of myocyte mitophagy

  • Balance of metabolic and anti-oxidant effects in heart failure models

• Neurological Disease Applications:

  • Therapeutic enhancement of TIGAR's neuroprotective effects against ischemic brain damage

  • Exploitation of PPP activation to counter oxidative stress in neurodegenerative diseases

  • Investigation of TIGAR in traumatic brain injury and stroke models

• Metabolic Disorder Strategies:

  • Exploration of TIGAR's role in insulin resistance and diabetes

  • Assessment of potential in non-alcoholic fatty liver disease

  • Investigation of interactions with other metabolic regulators

• Aging and Cellular Senescence:

  • Examination of TIGAR in longevity pathways

  • Connection to cellular senescence mechanisms

  • Potential for rejuvenation strategies through metabolic reprogramming

Research has established TIGAR as a multifunctional protein with diverse roles in cellular homeostasis and stress responses. The development of specific modulators of TIGAR activity, combined with precise understanding of context-dependent functions, holds significant promise for therapeutic applications across multiple disease states .