C12ORF5 Human, TAT
Description
Molecular Structure and Production
C12ORF5 Human, TAT consists of the full-length 270-amino-acid TIGAR protein fused to a 13-residue TAT peptide (GRKKRRQRRRPPQ) at the C-terminus . Key structural and production details include:
The TAT peptide enables efficient cellular uptake without requiring transfection agents, making it valuable for in vitro and in vivo studies .
Biochemical Mechanism
TIGAR functions as a fructose-2,6-bisphosphatase, hydrolyzing fructose-2,6-bisphosphate (F2,6BP) into fructose-6-phosphate (F6P) . This activity:
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Inhibits glycolysis by reducing F2,6BP, a potent activator of phosphofructokinase-1 (PFK-1) .
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Promotes the PPP, increasing NADPH production to scavenge reactive oxygen species (ROS) .
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Supports cell survival under stress by balancing ATP synthesis and antioxidant defense .
In lymphocytes, TIGAR induction via the PI3K/AKT pathway enhances PPP flux, reducing ROS and inhibiting autophagy during activation .
Neuroprotection in Hypoglycemia
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In vivo studies in transgenic mice overexpressing TIGAR demonstrated preserved blood-brain barrier (BBB) integrity during hypoglycemia, with reduced claudin-5 degradation and vascular permeability .
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In human brain microvascular endothelial cells (HBMECs), TIGAR-TAT pretreatment (0.1–5.0 µg/mL) prevented tight junction disruption under low-glucose conditions, maintaining NADPH levels and PPP flux .
Cancer Cell Survival
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TIGAR overexpression in glioma cells enhanced mitochondrial respiration and ROS scavenging, promoting survival under hypoxia .
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In cervical carcinoma cells, TIGAR knockdown increased ROS and sensitized cells to radiation-induced apoptosis .
Autophagy Regulation
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TIGAR suppresses autophagy by elevating NADPH and reducing oxidative stress. Silencing TIGAR in lymphocytes increased autophagy markers (e.g., reduced p62 levels) .
Therapeutic Implications
Experimental Use Guidelines
Product Specs
Repeated freeze-thaw cycles should be avoided to maintain protein stability.
(a) Analysis by RP-HPLC (Reverse Phase High-Performance Liquid Chromatography).
(b) Analysis by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).
Q&A
What is C12ORF5 and how does it relate to TIGAR?
C12ORF5 (chromosome 12 open reading frame 5) is the gene name for the protein known as TIGAR (TP53-induced glycolysis and apoptosis regulator). This 270 amino acid protein is induced by the p53 tumor suppressor pathway and functions to protect cells against oxidative stress . The protein contains a bisphosphate domain with similarity to the glycolytic enzyme that degrades fructose-2,6-bisphosphate, which gives it its functional properties .
What are the key functions of TIGAR in cellular metabolism?
TIGAR serves as a negative regulator of glycolysis by functioning as a fructose-2,6-bisphosphatase. By hydrolyzing fructose-2,6-bisphosphate, a key activator of glycolysis, TIGAR redirects glucose metabolism from glycolysis to the pentose phosphate pathway (PPP) . This metabolic shift results in:
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Increased NADPH production, enhancing cellular antioxidant capacity
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Reduced reactive oxygen species (ROS) levels
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Improved pentose phosphate pathway flux
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Protection against oxidative stress-induced apoptosis
These functions allow TIGAR to modulate the apoptotic response to p53, enabling cells to survive mild or transient stresses .
What is the significance of TAT fusion in recombinant C12ORF5/TIGAR proteins?
The TAT domain (derived from HIV Trans-Activator of Transcription protein) is a cell-penetrating peptide that facilitates protein transduction across cell membranes. When fused to C12ORF5/TIGAR:
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It enables efficient cellular uptake of the recombinant protein
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It allows direct delivery of functional TIGAR protein to cells
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It bypasses the need for gene transfection or viral transduction
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It permits rapid experimental manipulation of TIGAR levels
Commercial recombinant versions, such as those described in the search results, typically contain a 13-residue C-terminal peptide incorporating the TAT transduction domain (sequence: YGRKKRRQRRR) .
How should recombinant C12ORF5/TIGAR proteins be stored and handled?
Based on manufacturer recommendations:
| Storage Condition | Duration | Product Form |
|---|---|---|
| 4°C | 2-4 weeks (if entire vial will be used) | Solution |
| -20°C | Longer periods | Solution |
| -20°C to -70°C | 6 months from receipt (as supplied) | Filtered solution |
| -20°C to -70°C | 3 months (under sterile conditions after opening) | Filtered solution |
For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) and avoid multiple freeze-thaw cycles .
How can I verify TIGAR/C12ORF5 activity in experimental settings?
TIGAR activity can be measured by its ability to cleave p-Nitrophenyl phosphate (pNPP). A standard activity assay protocol involves:
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Preparing reactions with assay buffer (50 mM Tris, pH 7.5)
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Adding recombinant TIGAR protein (typically 2 μg per well)
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Adding substrate (1.25 mM)
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Incubating at appropriate temperature/time
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Stopping reactions with 0.2 M NaOH
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Measuring absorbance at 410 nm
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Calculating specific activity using the formula:
Specific Activity (pmol/min/μg) = Adjusted Absorbance (OD) × Conversion Factor (pmol/OD) / [Incubation time (min) × amount of enzyme (μg)]
The specific activity should be >10 pmol/min/μg under standard conditions .
What detection methods are suitable for analyzing TIGAR/C12ORF5 expression?
Several validated methods for detecting TIGAR include:
Western Blot Analysis:
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HeLa, Jurkat, and U2OS cell lines show detectable endogenous TIGAR expression
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TIGAR appears as a specific band at approximately 30 kDa under reducing conditions
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Recommended antibody dilution: 0.5 μg/mL for primary antibody
Immunofluorescence:
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TIGAR can be detected in fixed cells using appropriate antibodies
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Localization is primarily cytoplasmic and nuclear
Immunohistochemistry:
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TIGAR is detectable in paraffin-embedded tissue sections
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Shows localization to cytoplasm and nuclei in cancer tissues
How does TIGAR protect against hypoglycemic stress in brain endothelial cells?
Research indicates that TIGAR plays a critical role in maintaining brain microvessel integrity during hypoglycemic stress. Transgenic mice studies have shown that:
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Microvessel integrity was dramatically compromised (59.41% of wild-type mice) in TIGAR transgenic mice stressed by hypoglycemia
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Melatonin (a potent antioxidant) at 400 nmol/L protected against hypoglycemic stress-induced brain endothelial tight junction injury
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TIGAR couples with calmodulin during low glucose conditions, promoting TIGAR tyrosine nitration
The mechanism involves TIGAR-dependent NADPH generation, which protects tight junctions in human brain microvascular endothelial cells. The Y92 residue is particularly critical, as mutation at this position decreases NADPH generation by 55.60% and abolishes the protective effect on tight junctions .
What is the significance of TIGAR's tyrosine nitration and interaction with calmodulin?
FRET (fluorescence resonance energy transfer) imaging studies revealed that:
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Under low glucose stress, TIGAR couples with calmodulin
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This interaction promotes TIGAR tyrosine nitration
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Tyrosine 92 appears to be a critical residue for this process
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Mutation of Y92 significantly impairs TIGAR-dependent NADPH generation (55.60% decrease)
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This mutation also eliminates TIGAR's protective effect on tight junctions in brain endothelial cells
This interaction represents a novel regulatory mechanism for TIGAR function under stress conditions and suggests potential therapeutic targets for protecting against hypoglycemic brain injury.
How does TIGAR contribute to cancer cell survival?
TIGAR plays multiple roles in cancer progression and survival:
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DNA Repair Promotion: TIGAR activates PPP flux in a CDK5-ATM-dependent signaling pathway during hypoxia and genome stress-induced DNA damage responses
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Metabolic Adaptation: TIGAR protects glioma cells from hypoxia and ROS-induced cell death by:
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Intestinal Tumor Progression: TIGAR is involved in promoting intestinal tumor growth and progression
These mechanisms make TIGAR a potential target for cancer therapies, particularly in contexts where metabolic vulnerabilities can be exploited.
What are the key structural features of recombinant C12ORF5/TIGAR proteins?
What expression systems and purification methods are used for recombinant TIGAR production?
Recombinant TIGAR/C12ORF5 is typically:
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Purified by:
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Formulated in buffers containing:
The final product is typically supplied as either a sterile filtered colorless solution or in lyophilized form, with purity >90-95% as determined by SDS-PAGE .
What factors might affect TIGAR activity in experimental settings?
Several factors can impact TIGAR enzymatic function:
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Buffer conditions: pH is critical for optimal activity (typically pH 7.5)
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Stability concerns: Multiple freeze-thaw cycles can reduce activity
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Protein modifications: Tyrosine nitration (especially at Y92) significantly affects function
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Cellular context: Hypoxia, oxidative stress, and metabolic state influence activity
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Interacting proteins: Calmodulin binding alters TIGAR function under stress conditions
When activity is lower than expected, consider using fresh protein preparations, optimizing buffer conditions, or adding stabilizing agents like DTT.
How can I determine if TIGAR/C12ORF5 is functioning properly in my cellular system?
To verify TIGAR function:
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Measure fructose-2,6-bisphosphate levels: Should decrease with active TIGAR
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Assess pentose phosphate pathway activity: Should increase (via NADPH production)
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Monitor ROS levels: Should decrease with functional TIGAR
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Evaluate cell survival under oxidative stress: TIGAR expression should confer protection
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Analyze tight junction integrity in endothelial models: TIGAR protects against disruption during hypoglycemic stress
Parallel experiments with Y92 mutant versions can serve as excellent negative controls for TIGAR-specific effects .
Product Science Overview
The C12ORF5 gene was discovered in the early 2000s and has since been studied extensively. The protein encoded by this gene is known as TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator). TIGAR is a newly discovered enzyme that largely regulates glucose breakdown in human cells . The protein has a tertiary structure similar to the histidine phosphatase fold and is approximately 30 kDa in size. The core of TIGAR is a β-sandwich composed of a six-stranded β-sheet encircled by four α-helices .
TIGAR plays a crucial role in cellular metabolism by regulating the levels of fructose-2,6-bisphosphate, a key regulator of glycolysis. By lowering the levels of fructose-2,6-bisphosphate, TIGAR inhibits glycolysis and promotes the pentose phosphate pathway, which is essential for generating NADPH and ribose-5-phosphate . This shift in metabolic pathways helps cells to manage oxidative stress and repair DNA damage.
Additionally, TIGAR has the ability to protect cells from apoptosis (programmed cell death) induced by oxidative stress. This protective function is particularly important in cancer cells, where TIGAR activity can contribute to tumor survival and growth .
The p53 tumor suppressor protein, which is activated in response to DNA damage, induces the expression of TIGAR. This connection between p53 and TIGAR has made the C12ORF5 gene a target of interest in cancer research. Studies have shown that TIGAR is overexpressed in various types of cancer, including breast, liver, and colorectal cancers . The overexpression of TIGAR in cancer cells helps them to survive under conditions of metabolic stress and contributes to their resistance to chemotherapy.
Researchers are exploring the potential of targeting TIGAR as a therapeutic strategy for cancer treatment. By inhibiting TIGAR activity, it may be possible to sensitize cancer cells to chemotherapy and reduce their ability to survive under metabolic stress .
