PGK1 Human, Active

What is the basic function of PGK1 in human cellular metabolism?

PGK1 serves as an essential enzyme in the aerobic glycolysis pathway, where it catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP, producing 3-phosphoglycerate and ATP . Beyond its canonical role in glycolysis, PGK1 participates in multiple biological processes including angiogenesis, autophagy, and DNA repair . This multifunctional nature makes PGK1 a critical player in cellular energy production and various regulatory pathways across different cell types.

How is PGK1 expression regulated in normal vs. pathological states?

This upregulation is often driven by hypoxia-inducible factor 1-alpha (HIF-1α), which activates PGK1 transcription under hypoxic conditions common in tumor microenvironments. Additionally, the long non-coding RNA MVIH has been shown to repress PGK1 expression in HCC, affecting tumor-induced angiogenesis . Post-translational modifications, including phosphorylation, acetylation, and glycosylation, further modulate PGK1 activity in different pathological contexts.

What cellular pathways and biological processes involve PGK1?

PGK1 participates in multiple biological pathways beyond glycolysis:

• Apoptosis Regulation: PGK1 can inhibit apoptotic pathways, as demonstrated in neuronal survival mechanisms .

• Angiogenesis: Extracellular PGK1 suppresses angiogenesis by reducing VEGF levels and enhancing angiostatin generation in prostate cancer .

• Immunomodulation: PGK1 can reduce tumor growth in Lewis lung carcinoma by downregulating COX-2 expression and promoting anti-tumor immunity .

• Epithelial-Mesenchymal Transition (EMT): The HIF-1α/PGK1 axis mediates EMT, increasing metastatic potential in HCC and other cancers .

• Neurodegenerative Processes: PGK1 plays a central role in neuronal energy metabolism, with implications for Parkinson's disease pathogenesis .

This multifunctionality explains the diverse and sometimes contradictory roles of PGK1 in different disease contexts.

How does PGK1 contribute to cancer progression and what are its context-dependent roles?

PGK1 exhibits complex, context-dependent roles in cancer progression. Intracellularly, elevated PGK1 expression generally promotes tumor cell proliferation and metabolism. Analysis of TCGA data shows that patients with high PGK1 expression had a median survival of 3.1 years compared to 5.1 years in the low PGK1 expression group in HCC patients . This poor prognosis association has been validated in multiple cancer types including breast cancer, where PGK1 serves as a promising invasion promoter and survival biomarker .

Conversely, extracellular PGK1 can function as a tumor suppressor by inhibiting angiogenesis. PGK1 secreted into the cellular matrix reduces VEGF levels and enhances angiostatin generation, thereby suppressing tumor vascularization in prostate cancer . This dichotomy illustrates the complex nature of PGK1's role in cancer biology.

The metastatic capacity of cancer cells is also influenced by PGK1 through the HIF-1α/PGK1-mediated epithelial-mesenchymal transition (EMT). Higher expression levels of PGK1 and HIF-1α are detectable in highly metastatic HCC cells (HCCLM9) compared to less metastatic MHCC97L cells . Similarly, upregulated HIF-1α and PGK1 activate the Notch pathway to promote progression of human glioblastoma .

What is the relationship between PGK1 and neurodegenerative diseases, particularly Parkinson's disease?

PGK1 has emerged as a central leverage point in Parkinson's disease (PD) pathophysiology. Contrary to initial expectations, PGK1 activity is a critical modulator of glycolytic throughput in neurons. Terazosin (TZ), which enhances PGK1 activity, confers significant neuroprotection in various PD models (mouse, rat, Drosophila, and human induced pluripotent stem cells) .

Clinical evidence supports this relationship. Retrospective analysis of patients using TZ for benign prostate hyperplasia showed that prolonged use reduced the risk of developing PD by approximately 37% compared to tamsulosin (which has the same molecular target but a different chemical structure) . More recent three-way analysis of different treatments confirmed TZ's beneficial effects in PD .

Genetically, PGK1 is part of the PARK12 susceptibility locus for PD, and certain PGK1 mutations in humans are characterized by early-onset PD . These findings suggest that enhanced PGK1 activity and increased glycolysis may alleviate neurodegeneration, potentially by addressing the energy deficiency that contributes to neuronal dysfunction and death in neurodegenerative diseases .

How do post-translational modifications affect PGK1 function and its role in disease?

Post-translational modifications (PTMs) profoundly influence PGK1's function and its role in disease. Although not explicitly detailed in the provided search results, research has established that PGK1 undergoes several important PTMs:

• Phosphorylation: Phosphorylation of PGK1 (particularly at serine residues) can alter its catalytic activity, subcellular localization, and interaction with other proteins.

• Acetylation: Acetylation of PGK1 can modulate its enzymatic activity and influence its non-glycolytic functions.

• Glycosylation: Glycosylation affects PGK1 stability and potentially its secretion into the extracellular environment.

These modifications are dynamically regulated in disease states. For example, in cancer cells with upregulated HIF-1α (such as leukemic K562 cells), PGK1 expression and activity are increased, protecting cells from reactive oxygen species (ROS) and apoptosis while enhancing glycolysis . Understanding the specific PTMs and their effects on PGK1 in different pathological contexts remains an important area for further research.

How can PGK1 activity be accurately measured in experimental settings?

Measuring PGK1 activity requires specialized techniques due to the rapid nature of the PGK-catalyzed reaction. A widely used approach is coupling the PGK-catalyzed reaction with the pyruvate dehydrogenase (GAPDH) reaction as follows:

• Coupled Enzyme Assay: Since the PGK self-catalyzed reaction is rapid and not easily detectable directly, researchers use an indirect approach by coupling the PGK-catalyzed reaction with the GAPDH-catalyzed reaction. GAPDH catalyzes the production of 1,3-BPG from glyceraldehyde phosphate .

• Spectrophotometric Detection: The product of the GAPDH forward reaction, NADH, has strong absorption at 339 nm. Thus, the strength of the PGK reaction can be monitored by measuring changes in NADH concentration spectrophotometrically .

• Reaction Components: A typical reaction mixture includes purified recombinant human PGK1 protein (2 μg/ml) mixed with substrates (1.6 mM GAP, 1 mM β-NAD, 1 mM ADP, 20 ng/μl GAPDH) in an appropriate buffer (20 mM Tris, 100 mM NaCl, 0.1 mM MgSO4) .

• Principle of Measurement: If PGK activity is enhanced (for example, by a potential activator), this causes a decrease in 1,3-BPG concentration in the reaction system, promoting the GAPDH forward reaction and increasing NADH production, which can be measured by absorbance .

This coupled enzyme assay provides a reliable and quantitative method for assessing PGK1 activity in experimental settings.

What approaches can be used to express and purify human PGK1 protein for in vitro studies?

Expression and purification of human PGK1 protein for in vitro studies typically follows these methodological steps:

• Cloning: The cDNA of human-derived PGK1 is cloned into an expression vector such as pET28a(+) . This vector typically includes a His-tag for easier purification.

• Expression System: The recombinant vector is transformed into bacterial expression systems, commonly BL21(DE3) chemically competent E. coli cells .

• Protein Expression Induction: Protein expression is induced using IPTG (isopropyl β-D-1-thiogalactopyranoside), typically at a concentration of 0.5 mM .

• Cell Lysis: After bacterial growth, cells are lysed in an extraction buffer containing components such as:

  • 20 mM sodium phosphate

  • 500 mM NaCl

  • 5 mM imidazole

  • 5% glycerol

  • Protease inhibitor cocktail

  • pH adjusted to approximately 7.9

• Purification: The supernatant is purified using affinity chromatography, typically with nickel beads for His-tagged proteins .

• Elution: The protein is eluted using an elution buffer containing higher imidazole concentration (around 80 mM) .

• Validation: The eluate is subjected to SDS-PAGE to verify the molecular mass and purity of the purified PGK1 protein .

This protocol yields pure, active human PGK1 protein suitable for enzymatic assays, structural studies, and other in vitro applications.

What experimental models are appropriate for studying PGK1's role in different diseases?

Several experimental models have proven valuable for investigating PGK1's role in various diseases:

For Cancer Research:

• Cell Lines: Liver cancer cell lines (SNU449, SNU182, HuH7, JHH5, HepG2, HCCLM3) show differential expression of PGK1 and can be used to study its role in hepatocellular carcinoma . HuH7 demonstrates the lowest protein expression levels of PGK1 and is suitable for overexpression assays, while the other cell lines with higher PGK1 expression are appropriate for knockdown experiments .

• Patient-Derived Samples: Analysis of PGK1 expression in clinical HCC tissues compared to normal tissue samples based on databases like TCGA and GTEx provides valuable insights into clinical relevance .

• Western Blotting and Immunohistochemistry: These techniques can be used to detect protein expression of PGK1 in various cancer cell lines and tissue samples .

For Neurodegenerative Disease Research:

• Multiple Model Approach: Combining different models provides more robust validation:

  • Drosophila oxidative stress model

  • Neuronal cell oxygen-glucose deprivation model

  • LPS-induced cellular inflammation model

• Animal Models: Mouse, rat, and Drosophila models have been effectively used to study PGK1's role in Parkinson's disease .

• Human iPSCs: Induced pluripotent stem cells provide a human-relevant system for studying PGK1 in neurodegeneration .

Computational Approaches:

• Virtual Screening: This technique can be used to search for potential PGK1 activators or inhibitors by screening compound libraries .

• Molecular Docking: Structural analysis of PGK1-ligand interactions helps in understanding binding modes and predicting functional effects .

This multi-model approach enables comprehensive investigation of PGK1's diverse roles in different disease contexts.

What is the current status of PGK1 as a biomarker in cancer diagnosis and prognosis?

PGK1 has emerged as a promising biomarker for cancer diagnosis and prognosis, particularly in hepatocellular carcinoma (HCC). Analysis of clinical data from TCGA and GTEx databases demonstrates that PGK1 expression levels are significantly elevated in HCC (n=365) compared to normal tissues (n=160) . This differential expression pattern makes PGK1 a potential diagnostic biomarker for early detection of HCC.

These findings collectively establish PGK1 as a valuable biomarker for early HCC diagnosis and prognosis assessment, with potential applications in clinical decision-making.

What advances have been made in developing PGK1 activators for therapeutic purposes?

Significant progress has been made in identifying and developing PGK1 activators for therapeutic applications, particularly for neurodegenerative disorders:

• Virtual Screening Approaches: Researchers have employed virtual screening techniques to search for potential apoptosis inhibitors targeting PGK1. This computational approach involves screening natural compound libraries to identify molecules that can enhance PGK1 activity .

• Identified Activators: Several compounds have been identified as PGK1 activators, including:

  • 7979989

  • Z112553128

  • AK-693/21087020

  These compounds demonstrated activation of PGK1 within specific concentration ranges (10 μM-1 nM for 7979989 and Z112553128; 1 μM-1 nM for AK-693/21087020) . Interestingly, AK-693/21087020 showed inhibitory effects at higher concentrations (10 μM), suggesting dose-dependent mechanisms.

• Binding Mode Analysis: Molecular docking studies have elucidated the binding modes of these compounds to PGK1, explaining their differential effects on enzyme activity. Compounds with similar molecular backbones (7979989 and Z112553128) produced similar interactions with surrounding amino acid residues, resulting in comparable activity profiles .

• Repurposed Medications: Terazosin (TZ), a medication traditionally used for benign prostate hyperplasia, has been identified as a PGK1 activator with neuroprotective effects in multiple Parkinson's disease models . This discovery has led to retrospective clinical analyses showing reduced PD risk in patients taking TZ long-term .

These advances represent promising steps toward developing PGK1-targeted therapeutics for neurodegenerative disorders and potentially other conditions where enhanced glycolytic activity would be beneficial.

How does PGK1 function in different cellular compartments and what are the implications for targeting PGK1?

PGK1 exhibits distinct functions depending on its cellular localization, which has important implications for therapeutic targeting:

• Cytosolic PGK1: In the cytoplasm, PGK1 primarily functions as a glycolytic enzyme, catalyzing the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate while generating ATP . This metabolic role supports cellular energy production, particularly important in highly energetic cells like neurons and cancer cells.

• Nuclear PGK1: When localized to the nucleus, PGK1 can participate in DNA replication and repair processes. Although not explicitly detailed in the provided search results, research has shown that nuclear PGK1 can function as a protein kinase that phosphorylates proteins involved in DNA replication and repair.

• Extracellular PGK1: When secreted into the extracellular matrix, PGK1 displays anti-angiogenic properties. It reduces VEGF levels and enhances the generation of angiostatin, thereby inhibiting angiogenesis in prostate cancer . The long non-coding RNA MVIH can repress PGK1 secretion, activating tumor-induced angiogenesis in hepatocellular carcinoma .

• Mitochondrial PGK1: PGK1 can be translocated to mitochondria under certain conditions, where it may interact with mitochondrial proteins and influence energy metabolism.

This compartment-specific functionality suggests that therapeutic strategies targeting PGK1 should consider:

• Localization-Specific Targeting: Developing approaches that modulate PGK1 activity in specific cellular compartments rather than globally.

• Context-Dependent Interventions: Enhancing intracellular PGK1 may benefit neurodegenerative conditions by boosting energy production, while inhibiting it might be beneficial in certain cancers.

• Dual-Action Strategies: In cancer therapy, inhibiting intracellular PGK1 while promoting its extracellular secretion could potentially target both cancer metabolism and angiogenesis simultaneously.

Understanding these compartment-specific functions is crucial for developing precision-medicine approaches centered on PGK1 modulation.

What are the key methodological challenges in studying PGK1 and how can they be addressed?

Studying PGK1 presents several methodological challenges that researchers must navigate:

• Rapid Reaction Kinetics: The PGK self-catalyzed reaction is rapid and not easily detectable directly.

  • Solution: Use indirect coupled enzyme assays with GAPDH, where the production of NADH can be monitored spectrophotometrically at 339 nm . This approach provides a reliable readout of PGK1 activity.

• Protein Stability and Activity: Maintaining PGK1 stability and activity during purification and experimental procedures.

  • Solution: Include glycerol (typically 5%) in extraction buffers and protease inhibitor cocktails to preserve protein integrity . Optimize buffer conditions (pH, salt concentration) based on specific experimental requirements.

• Distinguishing Isoforms: PGK has different isoforms with potentially distinct functions.

  • Solution: Use isoform-specific antibodies for western blotting and immunohistochemistry. Employ recombinant expression of specific isoforms for functional studies.

• Context-Dependent Activities: PGK1 exhibits different functions depending on cellular location and disease context.

  • Solution: Use cell fractionation techniques to study compartment-specific activities. Employ multiple disease models to validate findings across different contexts.

• Translating In Vitro Findings: Bridging the gap between in vitro enzymatic studies and in vivo disease relevance.

  • Solution: Implement a multi-model approach combining in vitro assays, cell culture systems, animal models, and clinical sample analysis to establish robust translational relevance .

By addressing these methodological challenges with appropriate technical solutions, researchers can enhance the quality and translational value of PGK1-focused studies.

What are the most promising future research avenues for PGK1-targeted therapeutics?

Several promising research directions could advance PGK1-targeted therapeutics:

• Selective Activators for Neurodegenerative Diseases: Further development of selective PGK1 activators could yield novel therapeutics for Parkinson's disease and potentially other neurodegenerative conditions . Structure-activity relationship studies of compounds like terazosin and the recently identified activators (7979989, Z112553128, AK-693/21087020) could guide the design of more potent and selective molecules .

• Dual-Action Cancer Therapeutics: Developing strategies that simultaneously inhibit intracellular PGK1 (to decrease tumor cell proliferation) while promoting extracellular PGK1 (to inhibit angiogenesis) could provide novel cancer treatment approaches .

• Biomarker Development and Validation: Further validation of PGK1 as a diagnostic and prognostic biomarker across different cancer types, with standardization of assessment methods for clinical implementation .

• Compartment-Specific Targeting: Creating therapeutic approaches that selectively modulate PGK1 activity in specific cellular compartments (cytosol, nucleus, mitochondria, extracellular space) to achieve desired effects while minimizing off-target consequences.

• Combination Therapies: Investigating synergistic effects of PGK1 modulators with existing therapeutics, such as chemotherapy in cancer or dopaminergic agents in Parkinson's disease.

• Personalized Medicine Approaches: Developing methods to stratify patients based on PGK1 expression or activity profiles to guide selection of appropriate therapeutic interventions.

These research directions could significantly advance our understanding of PGK1 biology and lead to novel therapeutic strategies for managing cancer, neurodegenerative diseases, and potentially other conditions involving metabolic dysregulation.