The long noncoding RNA glycoLINC assembles a lower glycolytic metabolon to promote glycolysis.

Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.

PTEN Suppresses Glycolysis by Dephosphorylating and Inhibiting Autophosphorylated PGK1.

The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly regulates glycolysis in tumor cells is unclear. We demonstrate here that PTEN directly interacts with phosphoglycerate kinase 1 (PGK1). PGK1 functions not only as a glycolytic enzyme but also as a protein kinase intermolecularly autophosphorylating itself at Y324 for activation. The protein phosphatase activity of PTEN dephosphorylates and inhibits autophosphorylated PGK1, thereby inhibiting glycolysis, ATP production, and brain tumor cell proliferation. In addition, knockin expression of a PGK1 Y324F mutant inhibits brain tumor formation. Analyses of human glioblastoma specimens reveals that PGK1 Y324 phosphorylation levels inversely correlate with PTEN expression status and are positively associated with poor prognosis in glioblastoma patients. This work highlights the instrumental role of PGK1 autophosphorylation in its activation and PTEN protein phosphatase activity in governing glycolysis and tumorigenesis.

A model for stimulation of enzyme activity by a competitive inhibitor based on the interaction of terazosin and phosphoglycerate kinase 1.

The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.

Nuclear PGK1 Alleviates ADP-Dependent Inhibition of CDC7 to Promote DNA Replication.

DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development.

Macrophage-Associated PGK1 Phosphorylation Promotes Aerobic Glycolysis and Tumorigenesis.

Macrophages are a dominant leukocyte population in the tumor microenvironment and actively promote cancer progression. However, the molecular mechanism underlying the role of macrophages remains poorly understood. Here we show that polarized M2 macrophages enhance 3-phosphoinositide-dependent protein kinase 1 (PDPK1)-mediated phosphoglycerate kinase 1 (PGK1) threonine (T) 243 phosphorylation in tumor cells by secreting interleukin-6 (IL-6). This phosphorylation facilitates a PGK1-catalyzed reaction toward glycolysis by altering substrate affinity. Inhibition of PGK1 T243 phosphorylation or PDPK1 in tumor cells or neutralization of macrophage-derived IL-6 abrogates macrophage-promoted glycolysis, proliferation, and tumorigenesis. In addition, PGK1 T243 phosphorylation correlates with PDPK1 activation, IL-6 expression, and macrophage infiltration in human glioblastoma multiforme (GBM). Moreover, PGK1 T243 phosphorylation also correlates with malignance and prognosis of human GBM. Our findings demonstrate a novel mechanism of macrophage-promoted tumor growth by regulating tumor cell metabolism, implicating the therapeutic potential to disrupt the connection between macrophages and tumor cells by inhibiting PGK1 phosphorylation.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Mitochondrial translation deficiency impairs NAD+ -mediated lysosomal acidification.

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

Identification of the Most Immunoreactive Antigens of Candida auris to IgGs from Systemic Infections in Mice.

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.

The mutual interaction of glycolytic enzymes and RNA in post-transcriptional regulation.

About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term "moonlighting functions" was proposed. Recently developed advanced technologies in the field of RNA interactome capture now unveil the unexpected RNA binding activity of many metabolic enzymes, as exemplified here for the enzymes of glycolysis. Although for most of these proteins a precise binding mechanism, binding conditions, and physiological relevance of the binding events still await in-depth clarification, several well explored examples demonstrate that metabolic enzymes hold crucial functions in post-transcriptional regulation of protein synthesis. This widely conserved RNA-binding function of glycolytic enzymes plays major roles in controlling cell activities. The best explored examples are glyceraldehyde 3-phosphate dehydrogenase, enolase, phosphoglycerate kinase, and pyruvate kinase. This review summarizes current knowledge about the RNA-binding activity of the ten core enzymes of glycolysis in plant, yeast, and animal cells, its regulation and physiological relevance. Apparently, a tight bidirectional regulation connects core metabolism and RNA biology, forcing us to rethink long established functional singularities.

CircGLIS3 promotes gastric cancer progression by regulating the miR-1343-3p/PGK1 pathway and inhibiting vimentin phosphorylation.

BACKGROUND: Circular RNAs (circRNAs) have been proved to play crucial roles in the development of various cancers. However, the molecular mechanism of circGLIS3 involved in gastric cancer (GC) tumorigenesis has not been elucidated. METHODS: The higher expression level of circGLIS3 was identified in GC through RNA sequencing and subsequent tissue verification using Quantitative real-time PCR (qRT-PCR). A series of functional experiments in vitro and in vivo were performed to evaluated the effects of circGLIS3 on tumor growth and metastasis in GC. The interaction and regulation of circGLIS3/miR-1343-3p/PGK1 axis was confirmed by RNA pulldown, western blot, and rescue experiments. RIP and western blot were performed to demonstrate the role of circGLIS3 in regulating phosphorylation of VIMENTIN. We then used qRT-PCR and co culture system to trace circGLIS3 transmission via exosomal communication and identify the effect of exosomal circGLIS3 on gastric cancer and macrophages. Finally, RIP experiments were used to determine that EIF4A3 regulates circGLIS3 expression. RESULTS: CircGLIS3(hsa_circ_0002874) was significantly upregulated in GC tissues and high circGLIS3 expression was associated with advanced TNM stage and lymph node metastasis in GC patients. We discovered that overexpression of circGLIS3 promoted GC cell proliferation, migration, invasion in vitro and in vivo, while suppression of circGLIS3 exhibited the opposite effect. Mechanistically, circGLIS3 could sponge miR-1343-3p and up-regulate the expression of PGK1 to promote GC tumorigenesis. We also found that circGLIS3 reduced the phosphorylation of VIMENTIN at ser 83 site by binding with VIMENTIN. Moreover, it was proven that exosomal circGLIS3 could promote gastric cancer metastasis and the M2 type polarization of macrophages. In the final step, the mechanism of EIF4A3 regulating the generation of circGLIS3 was determined. CONCLUSION: Our findings demonstrate that circGLIS3 promotes GC progression through sponging miR-1343-3p and regulating VIMENTIN phosphorylation. CircGLIS3 is a potential therapeutic target for GC patients.

Glycolytic enzyme PGK1 promotes M1 macrophage polarization and induces pyroptosis of acute lung injury via regulation of NLRP3.

Acute lung injury (ALI) is characterized by an unregulated inflammatory reaction, often leading to severe morbidity and ultimately death. Excessive inflammation caused by M1 macrophage polarization and pyroptosis has been revealed to have a critical role in ALI. Recent study suggests that glycolytic reprogramming is important in the regulation of macrophage polarization and pyroptosis. However, the particular processes underlying ALI have yet to be identified. In this study, we established a Lipopolysaccharide(LPS)-induced ALI model and demonstrated that blocking glycolysis by using 2-Deoxy-D-glucose(2-DG) significantly downregulated the expression of M1 macrophage markers and pyroptosis-related genes, which was consistent with the in vitro results. Furthermore, our research has revealed that Phosphoglycerate Kinase 1(PGK1), an essential enzyme in the glycolysis pathway, interacts with NOD-, LRR- and pyrin domain-containing protein 3(NLRP3). We discovered that LPS stimulation improves the combination of PGK1 and NLRP3 both in vivo and in vitro. Interestingly, the absence of PGK1 reduces the phosphorylation level of NLRP3. Based on in vitro studies with mice bone marrow-derived macrophages (BMDMs), we further confirmed that siPGK1 plays a protective role by inhibiting macrophage pyroptosis and M1 macrophage polarization. The PGK1 inhibitor NG52 suppresses the occurrence of excessive inflammation in ALI. In general, it is plausible to consider a therapeutic strategy that focuses on modulating the relationship between PGK1 and NLRP3 as a means to mitigate the activation of inflammatory macrophages in ALI.

Acetylation of PGK1 at lysine 323 promotes glycolysis, cell proliferation, and metastasis in luminal A breast cancer cells.

BACKGROUND: In prior research employing iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) technology, we identified a range of proteins in breast cancer tissues exhibiting high levels of acetylation. Despite this advancement, the specific functions and implications of these acetylated proteins in the context of cancer biology have yet to be elucidated. This study aims to systematically investigate the functional roles of these acetylated proteins with the objective of identifying potential therapeutic targets within breast cancer pathophysiology. METHODS: Acetylated targets were identified through bioinformatics, with their expression and acetylation subsequently confirmed. Proteomic analysis and validation studies identified potential acetyltransferases and deacetylases. We evaluated metabolic functions via assays for catalytic activity, glucose consumption, ATP levels, and lactate production. Cell proliferation and metastasis were assessed through viability, cycle analysis, clonogenic assays, PCNA uptake, wound healing, Transwell assays, and MMP/EMT marker detection. RESULTS: Acetylated proteins in breast cancer were primarily involved in metabolism, significantly impacting glycolysis and the tricarboxylic acid cycle. Notably, PGK1 showed the highest acetylation at lysine 323 and exhibited increased expression and acetylation across breast cancer tissues, particularly in T47D and MCF-7 cells. Notably, 18 varieties acetyltransferases or deacetylases were identified in T47D cells, among which p300 and Sirtuin3 were validated for their interaction with PGK1. Acetylation at 323 K enhanced PGK1's metabolic role by boosting its activity, glucose uptake, ATP production, and lactate output. This modification also promoted cell proliferation, as evidenced by increased viability, S phase ratio, clonality, and PCNA levels. Furthermore, PGK1-323 K acetylation facilitated metastasis, improving wound healing, cell invasion, and upregulating MMP2, MMP9, N-cadherin, and Vimentin while downregulating E-cadherin. CONCLUSION: PGK1-323 K acetylation was significantly elevated in T47D and MCF-7 luminal A breast cancer cells and this acetylation could be regulated by p300 and Sirtuin3. PGK1-323 K acetylation promoted cell glycolysis, proliferation, and metastasis, highlighting novel epigenetic targets for breast cancer therapy.

Novel energy optimizer, meldonium, rapidly restores acute hypobaric hypoxia-induced brain injury by targeting phosphoglycerate kinase 1.

BACKGROUND: Acute hypobaric hypoxia-induced brain injury has been a challenge in the health management of mountaineers; therefore, new neuroprotective agents are urgently required. Meldonium, a well-known cardioprotective drug, has been reported to have neuroprotective effects. However, the relevant mechanisms have not been elucidated. We hypothesized that meldonium may play a potentially novel role in hypobaric hypoxia cerebral injury. METHODS: We initially evaluated the neuroprotection efficacy of meldonium against acute hypoxia in mice and primary hippocampal neurons. The potential molecular targets of meldonium were screened using drug-target binding Huprot™ microarray chip and mass spectrometry analyses after which they were validated with surface plasmon resonance (SPR), molecular docking, and pull-down assay. The functional effects of such binding were explored through gene knockdown and overexpression. RESULTS: The study clearly shows that pretreatment with meldonium rapidly attenuates neuronal pathological damage, cerebral blood flow changes, and mitochondrial damage and its cascade response to oxidative stress injury, thereby improving survival rates in mice brain and primary hippocampal neurons, revealing the remarkable pharmacological efficacy of meldonium in acute high-altitude brain injury. On the one hand, we confirmed that meldonium directly interacts with phosphoglycerate kinase 1 (PGK1) to promote its activity, which improved glycolysis and pyruvate metabolism to promote ATP production. On the other hand, meldonium also ameliorates mitochondrial damage by PGK1 translocating to mitochondria under acute hypoxia to regulate the activity of TNF receptor-associated protein 1 (TRAP1) molecular chaperones. CONCLUSION: These results further explain the mechanism of meldonium as an energy optimizer and provide a strategy for preventing acute hypobaric hypoxia brain injury at high altitudes.

METTL3 facilitates prostate cancer progression via inducing HOXC6 m6A modification and stabilizing its expression through IGF2BP2-dependent mechanisms.

HOXC6 (Homeobox C6) and methyltransferase-like 3 (METTL3) have been shown to be involved in the progression of prostate cancer (PCa). However, whether HOXC6 performs oncogenic effects in PCa via METTL3-mediated N6-methyladenosine (m6A) modification is not yet reported. The Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, scratch, sphere formation assays were applied for cell growth, invasion, migration and stemness analyses. Glycolysis was evaluated by measuring glucose consumption, lactate generation and ATP/ADP ratio. The N6-methyladenine (m6A) modification profile was determined by RNA immunoprecipitation (Me-RIP) assay. The proteins that interact with PGK1 (phosphoglycerate kinase 1) were confirmed by Co-immunoprecipitation assay. Tumor formation experiments in mice were conducted for in vivo assay. PCa tissues and cells showed highly expressed HOXC6 and METTL3. Functionally, the silencing of HOXC6 or METTL3 suppresses PCa cell proliferation, invasion, migration, stemness, and glycolysis. Moreover, METTL3-induced HOXC6 m6A modification to stabilize its expression. In addition, the m6A reader IGF2BP2 directly recognized and bound to HOXC6 mRNA, and maintained its stability, and was involved in the regulation of HOXC6 expression by METTL3. Furthermore, IGF2BP2 knockdown impaired PCa cell proliferation, invasion, migration, stemness, and glycolysis by regulating HOXC6. Besides that HOXC6 interacted with the glycoytic enzyme PGK1 in PCa cells. In vivo assays further showed that METTL3 silencing reduced the expression of HOXC6 and PGK1, and impeded PCa growth. METTL3 promoted PCa progression by maintaining HOXC6 expression in an m6A-IGF2BP2-dependent mechanism.

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.

Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis.

It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis.

Circ_0027446 promotes malignant development of glioblastoma by interacting with miR-346 to up-regulate PGK1.

Circular RNAs (circRNAs) can play essential roles in tumor development, including glioblastoma (GBM). The current study was performed to explore the function and mechanism of circ_0027446 in GBM progression. Circ_0027446, microRNA-346 (miR-346) and Phosphoglycerate kinase 1 (PGK1) levels were detected using reverse transcription-quantitative polymerase chain reaction assay. Cell behaviors were examined using Cell Counting Kit-8 assay, colony formation assay, EdU assay, flow cytometry, and transwell assay. Glycolytic metabolism was analyzed by commercial kits. The protein level was determined via western blot. The target interaction was analyzed by dual-luciferase reporter assay. Circ_0027446 function in vivo was explored by tumor xenograft assay. Circ_0027446 expression was significantly up-regulated in GBM samples and cells. Circ_0027446 down-regulation suppressed proliferation, invasion, glycolytic metabolism and enhanced apoptosis of GBM cells. MiR-346 was a target of circ_0027446, and circ_0027446 promoted GBM progression by sponging miR-346. PGK1 acted as a target gene of miR-346, and circ_0027446 interacted with miR-346 to regulate PGK1 expression. Overexpression of miR-346 inhibited malignant behaviors of GBM cells through down-regulating PGK1. Circ_0027446 contributed to tumor growth in vivo via miR-346/PGK1 axis. The current evidences demonstrated that circ_0027446 facilitated malignant progression of GBM through binding to miR-346 to up-regulate PGK1.

Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase.

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1.

BACKGROUND: The regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis. RESULTS: Our findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation. CONCLUSIONS: The posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.