C12ORF5 Human

What is C12ORF5 and what are its alternative names in scientific literature?

C12ORF5 is the gene encoding for a 270 amino acid protein known as TP53-induced glycolysis and apoptosis regulator (TIGAR). It is also referred to as probable fructose-2,6-bisphosphatase TIGAR in some publications. The gene was first discovered in 2005 by Kuang-Yu Jen and Vivian G. Cheung during computer-based searches for novel p53-regulated genes activated in response to ionizing radiation . The protein was later characterized in 2006 by Bensad who cloned and identified the c12orf5 gene and officially named it TIGAR .

What is the genomic location and structure of the C12ORF5 gene?

In humans, the C12ORF5 gene is located on chromosome 12p13-3 and consists of 6 exons . The complete C12orf5 mRNA is 8237 base pairs in length . The 12p13.32 region that includes this gene is paralogous to the 11q13.3 region . According to genetic databases, the gene is assigned several external identifiers including HGNC: 1185, NCBI Gene: 57103, Ensembl: ENSG00000078237, OMIM®: 610775, and UniProtKB/Swiss-Prot: Q9NQ88 .

What is the primary function of the TIGAR protein?

TIGAR functions primarily as a regulator of glucose metabolism with activity similar to fructose-2,6-bisphosphatase (Fru-2,6-BPase), a key enzyme in regulating glycolysis . Specifically, TIGAR blocks glycolysis and redirects glucose metabolism through the pentose phosphate pathway (PPP), which results in decreased intracellular concentration of reactive oxygen species (ROS) . This metabolic shift protects cells from oxidative stress and provides some protection from DNA damage-induced apoptosis . TIGAR enables cells to respond to mild or transient stresses by modulating the apoptotic response to p53, thus promoting cell survival under certain conditions .

How is TIGAR protein expression regulated?

• p53-dependent regulation: TIGAR is induced by the p53 pathway in response to cellular stress or DNA damage .

• p53-independent regulation: SP1 can participate in forming a DNA-protein complex to activate the TIGAR promoter in a p53-independent manner .

• CREB-mediated regulation: A cAMP response element (CRE) site in the −4 to +13 region of the TIGAR promoter can be recognized and bound by CREB, which functions as a transcription factor to regulate TIGAR expression .

• Hypoxia-induced regulation: Under hypoxic conditions, HIF1α promotes TIGAR expression by binding to the HIF response element (HRE) in the TIGAR promoter .

• NF-κB-dependent regulation: In the absence of oxygen-sensing prolyl hydroxylase-domain protein 1 (PHD1), NF-κB-dependent but HIF1α-independent signals can increase TIGAR transcription .

What is the role of TIGAR in cancer biology and therapeutic resistance?

TIGAR plays a complex role in cancer biology with evidence of both tumor-promoting and potentially tumor-suppressing activities depending on the context:

How does TIGAR influence metabolic reprogramming in normal and disease states?

TIGAR's influence on metabolic reprogramming extends beyond its basic function as a glycolysis regulator:

• Redirection to pentose phosphate pathway: TIGAR inhibits glycolysis and redirects glucose flux through the pentose phosphate pathway, which generates NADPH for antioxidant defense and ribose-5-phosphate for nucleotide synthesis .

• Mitochondrial localization and function: During exhaustive exercise, TIGAR can translocate to mitochondria to interact with ATP5A1 and promote mitochondrial production through the SIRT1/PGC1α axis, improving exercise tolerance, fatigue resistance, and delaying aging in mice .

• Neuroprotection: TIGAR plays a neuroprotective role against ischemic brain damage by enhancing pentose phosphate pathway flux and preserving mitochondrial functions .

• Response to hypoxia: Under hypoxic conditions, TIGAR can promote cancer cell survival by activating mitochondrial energy metabolism and oxygen consumption .

• OXPHOS and glycolytic reprogramming: Research has identified TIGAR's role in oxidative phosphorylation (OXPHOS) and glycolytic metabolic reprogramming, though the complete mechanisms remain under investigation .

What are the optimal conditions for working with recombinant TIGAR protein in biochemical assays?

When working with recombinant TIGAR protein in biochemical assays, researchers should consider the following optimal conditions:

• Storage conditions: Store at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, store frozen at -20°C. For long-term storage, add a carrier protein (0.1% HSA or BSA) to maintain stability. Multiple freeze-thaw cycles should be avoided to prevent protein degradation .

• Buffer composition: The recommended buffer for TIGAR protein solution (0.5 mg/ml) contains 20mM Tris-HCl buffer (pH8.0), 0.2M NaCl, 2mM DTT, and 10% glycerol .

• Purity considerations: Research-grade TIGAR protein should have a purity greater than 90.0% as determined by SDS-PAGE analysis to ensure reliable experimental results .

• Activity preservation: As TIGAR is sensitive to oxidation, maintaining reducing conditions with DTT or similar agents is crucial for preserving enzymatic activity.

• Expression systems: Commercially available recombinant TIGAR is typically produced in Escherichia coli, which may have implications for post-translational modifications compared to mammalian-expressed protein .

What experimental approaches are most effective for studying TIGAR's role in cancer metabolism?

Several experimental approaches have proven effective for studying TIGAR's role in cancer metabolism:

• CRISPR/Cas9-based knockout studies: Genome-scale CRISPR knockout screens have been successfully used to identify TIGAR as a modifier of PARP inhibitor response in cancer cells .

• Metabolic flux analysis: Measuring changes in glycolytic flux and pentose phosphate pathway activity in response to TIGAR manipulation provides direct evidence of metabolic reprogramming.

• ROS measurement assays: Since TIGAR affects cellular redox status by influencing NADPH production, measuring ROS levels using fluorescent probes (e.g., DCFDA, MitoSOX) is valuable for assessing TIGAR's functional impact.

• Drug sensitivity assays: Testing cancer cell sensitivity to various therapeutic agents (particularly PARP inhibitors like olaparib) in TIGAR-modulated cells helps identify potential synergistic treatment approaches .

• Subcellular localization studies: Immunofluorescence and subcellular fractionation have revealed that TIGAR can relocate to different cellular compartments (ER, nucleus, mitochondria) under stress conditions, influencing its function .

• Patient sample analysis: Correlating TIGAR expression levels with clinical outcomes in cancer patients provides translational relevance to basic research findings .

What are the technical challenges in studying TIGAR translocation between cellular compartments?

Studying TIGAR translocation between cellular compartments presents several technical challenges:

• Temporal dynamics: TIGAR translocation can be a rapid and transient response to cellular stress, requiring time-course experiments with precise timing.

• Stimulus-specific responses: Different stress stimuli (hypoxia, ischemia-reperfusion, exhaustive exercise) may trigger translocation to different compartments (mitochondria, ER, nucleus), necessitating stimulus-specific experimental designs .

• Protein tagging considerations: Fluorescent protein tags may affect TIGAR's localization or function, requiring careful validation of tagged constructs against endogenous protein behavior.

• Subcellular fractionation purity: Clean separation of cellular compartments is essential but technically challenging when studying protein translocation between compartments.

• Live-cell imaging limitations: Capturing real-time translocation events requires sophisticated live-cell imaging with minimal phototoxicity, particularly challenging in hypoxic conditions.

• Interaction validation: Confirming functional interactions with compartment-specific partners (like ATP5A1 in mitochondria) requires techniques such as proximity ligation assays or co-immunoprecipitation with compartment-specific controls .

What are the unresolved questions regarding TIGAR's contribution to therapeutic resistance?

Several unresolved questions remain regarding TIGAR's contribution to therapeutic resistance:

• Mechanism of BRCA1 regulation: While TIGAR knockdown has been shown to enhance sensitivity to PARP inhibitors via downregulation of BRCA1 and the Fanconi anemia pathway, the precise molecular mechanism connecting TIGAR to BRCA1 expression remains unclear .

• Cancer type specificity: It remains uncertain whether TIGAR's role in therapeutic resistance is consistent across different cancer types or whether tissue-specific factors influence its impact.

• Biomarker potential: The utility of TIGAR expression as a predictive biomarker for PARP inhibitor response has not been fully evaluated in clinical settings.

• Combination therapy strategies: Optimal approaches for targeting TIGAR in combination with existing therapies (beyond PARP inhibitors) require further investigation.

• Resistance mechanisms: Whether cancer cells can develop resistance to TIGAR inhibition, and through what mechanisms, remains to be determined.

How does TIGAR function in non-cancer pathological contexts?

While much research on TIGAR has focused on cancer, several non-cancer pathological contexts warrant further investigation:

• Neurological disorders: TIGAR has been shown to play a neuroprotective role against ischemic brain damage, but its role in other neurological disorders like neurodegenerative diseases remains largely unexplored .

• Metabolic diseases: Given TIGAR's central role in glucose metabolism, its potential involvement in metabolic disorders such as diabetes requires further study.

• Aging-related pathologies: TIGAR's interaction with the SIRT1/PGC1α axis in improving exercise tolerance and delaying aging in mice suggests potential roles in age-related pathologies .

• Inflammatory conditions: The connection between TIGAR and NF-κB signaling hints at possible roles in inflammatory disorders that have not been fully characterized .

• Cardiac ischemia/reperfusion: Given TIGAR's protection against ischemia-reperfusion injury in neurons, similar protective effects might exist in cardiac tissue during myocardial infarction.

What novel therapeutic approaches might emerge from targeting TIGAR in disease states?

Several novel therapeutic approaches might emerge from targeting TIGAR:

• Small molecule inhibitors: Development of specific small molecule inhibitors of TIGAR's fructose-2,6-bisphosphatase activity could potentiate existing cancer therapies, particularly PARP inhibitors .

• Metabolic intervention combinations: Combining TIGAR inhibition with other metabolic interventions (e.g., glycolysis inhibitors) might create synthetic lethality in cancer cells dependent on specific metabolic pathways.

• Compartment-specific targeting: Developing strategies to inhibit TIGAR in specific cellular compartments (e.g., mitochondria-targeted inhibitors) might allow for more nuanced therapeutic approaches with fewer side effects.

• TIGAR mimetics for neuroprotection: For conditions where TIGAR has protective effects, such as neurological ischemia, development of TIGAR mimetics might provide therapeutic benefit .

• Gene therapy approaches: In conditions where TIGAR restoration might be beneficial, targeted gene therapy to restore TIGAR function in specific tissues could emerge as a therapeutic strategy.