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Cancer Medicine Jan 2023Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential...
BACKGROUND
Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer.
METHODS
We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer.
RESULTS
PEPCK-M was highly expressed in estrogen receptor-positive (ER ) breast cancers. Decreased cell proliferation and G /G arrest were induced in ER breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER breast cancer were attenuated by PCK2 knockdown.
CONCLUSION
PEPCK-M promotes proliferation and cell cycle progression in ER breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER breast cancer.
Topics: Humans; Female; Phosphoenolpyruvate; Receptors, Estrogen; Breast Neoplasms; Glutamine; Phosphoenolpyruvate Carboxykinase (ATP); Mitochondria; TOR Serine-Threonine Kinases; Mechanistic Target of Rapamycin Complex 1
PubMed: 35757841
DOI: 10.1002/cam4.4969 -
The Plant Journal : For Cell and... Jul 2022Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C plants have focused on photosynthetic PEPC,...
Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.
Topics: Edible Grain; Phosphoenolpyruvate Carboxylase; Salinity; Salt Stress; Sorghum
PubMed: 35488514
DOI: 10.1111/tpj.15789 -
Proceedings of the National Academy of... Aug 2021(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic...
(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.
Topics: Antitubercular Agents; Drug Resistance, Microbial; Drug Tolerance; Humans; Hypoxia; Mycobacterium tuberculosis; Phosphoenolpyruvate; Tuberculosis
PubMed: 34426499
DOI: 10.1073/pnas.2105800118 -
Plant Biotechnology Journal Mar 2021Introduction of a C photosynthetic mechanism into C crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially...
Introduction of a C photosynthetic mechanism into C crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. CO labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C proteins introduced thus far, a functional C pathway is achievable in rice.
Topics: Malate Dehydrogenase; Oryza; Phosphoenolpyruvate Carboxylase; Photosynthesis; Pyruvate, Orthophosphate Dikinase; Zea mays
PubMed: 33016576
DOI: 10.1111/pbi.13487 -
The Journal of Physical Chemistry. B Jul 2022The environmental condition is a critical regulation factor for protein behavior in solution. Several studies have shown that macromolecular crowders can modulate...
The environmental condition is a critical regulation factor for protein behavior in solution. Several studies have shown that macromolecular crowders can modulate protein structures, interactions, and functions. Recent publications described the regulation of specific interaction by macromolecular crowders. However, the other category of protein-protein interaction, namely, the transient interaction, is rarely investigated, especially from the perspective of protein structure to study transient interactions between proteins. Here, we used nuclear magnetic resonance and small-angle X-ray/neutron scattering methods to structurally investigate the ensemble of the protein complex in dilute buffer and crowded environments. Histidine phosphocarrier protein (HPr) and the N-terminal domain of enzyme I (EIN) are the important components of the bacterial phosphotransfer system. Our results show that the addition of Ficoll-70 promotes HPr molecules to form the encounter complex with EIN maintained by long-range electrostatic interaction. However, when macromolecular crowder BSA is used, the soft interaction between BSA and HPr perturbs the active site of HPr, driving HPr to form an encounter complex with EIN at the weakly charged interface. Our results indicate that different macromolecular crowders could influence transient EIN-HPr interaction through different mechanisms and provide new insights into protein-protein interaction regulation in native environments.
Topics: Bacterial Proteins; Catalytic Domain; Histidine; Macromolecular Substances; Phosphoenolpyruvate Sugar Phosphotransferase System
PubMed: 35731981
DOI: 10.1021/acs.jpcb.2c02713 -
The FEBS Journal Dec 2021Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for...
Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRAS upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.
Topics: Argininosuccinate Synthase; Aspartic Acid; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Humans; Intracellular Signaling Peptides and Proteins; Metabolomics; Mutation; Phosphoenolpyruvate Carboxykinase (ATP); Phosphoenolpyruvate Carboxykinase (GTP); Proteomics; Proto-Oncogene Proteins p21(ras); Signal Transduction
PubMed: 34227245
DOI: 10.1111/febs.16111 -
Diabetes Jun 2019Hepatocyte glucose production is a complex process that integrates cell-autonomous mechanisms with cellular signaling, enzyme activity modulation, and gene...
Hepatocyte glucose production is a complex process that integrates cell-autonomous mechanisms with cellular signaling, enzyme activity modulation, and gene transcription. Transcriptional mechanisms controlling glucose production are redundant and involve nuclear hormone receptors and unliganded transcription factors (TFs). Our knowledge of this circuitry is incomplete. Here we used DNA affinity purification followed by mass spectrometry to probe the network of hormone-regulated TFs by using phosphoenolpyruvate carboxykinase () and glucose-6-phosphatase () in liver and primary hepatocytes as model systems. The repertoire of insulin-regulated TFs is unexpectedly broad and diverse. Whereas in liver the two test promoters are regulated by largely overlapping sets of TFs, in primary hepatocytes and regulation diverges. Insulin treatment preferentially results in increased occupancy by the two promoters, consistent with a model in which the hormone's primary role is to recruit corepressors rather than to clear activators. Nine insulin-responsive TFs are present in both models, but only FoxK1, FoxA2, ZFP91, and ZHX3 require an intact insulin response sequence for binding. Knockdown of FoxK1 in primary hepatocytes decreased both glucose production and insulin's ability to suppress it. The findings expand the repertoire of insulin-dependent TFs and identify FoxK1 as a contributor to insulin signaling.
Topics: Animals; Forkhead Transcription Factors; Gene Expression Regulation; Gene Knockdown Techniques; Gluconeogenesis; Glucose-6-Phosphatase; Hepatocytes; Insulin; Intracellular Signaling Peptides and Proteins; Liver; Mice; Phosphoenolpyruvate Carboxykinase (GTP); Primary Cell Culture; Transcription Factors
PubMed: 30936148
DOI: 10.2337/db18-1236 -
Molecules (Basel, Switzerland) Jun 2024The phosphoenol pyruvate-oxaloacetate-pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol... (Review)
Review
The phosphoenol pyruvate-oxaloacetate-pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol pyruvate-oxaloacetate-pyruvate (POP) node is the switch point among the major metabolic pathways existing in most living organisms. POP-AAs have widespread applications in the nutrition, food, and pharmaceutical industries. These amino acids have been predominantly produced in and through microbial fermentation. With the rapid increase in market requirements, along with the global food shortage situation, the industrial production capacity of these two bacteria has encountered two bottlenecks: low product conversion efficiency and high cost of raw materials. Aiming to push forward the update and upgrade of engineered strains with higher yield and productivity, this paper presents a comprehensive summarization of the fundamental strategy of metabolic engineering techniques around phosphoenol pyruvate-oxaloacetate-pyruvate node for POP-AA production, including L-tryptophan, L-tyrosine, L-phenylalanine, L-valine, L-lysine, L-threonine, and L-isoleucine. Novel heterologous routes and regulation methods regarding the carbon flux redistribution in the POP node and the formation of amino acids should be taken into consideration to improve POP-AA production to approach maximum theoretical values. Furthermore, an outlook for future strategies of low-cost feedstock and energy utilization for developing amino acid overproducers is proposed.
Topics: Metabolic Engineering; Amino Acids; Oxaloacetic Acid; Escherichia coli; Phosphoenolpyruvate; Corynebacterium glutamicum; Pyruvic Acid; Metabolic Networks and Pathways; Fermentation
PubMed: 38930958
DOI: 10.3390/molecules29122893 -
Genes Jul 2020C photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C photosynthetic pathway have an...
C photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C and C species have elucidated the genetic diversity underpinning the C photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C gene families in an important C crop, (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10). Genetic diversity of C genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.
Topics: Domestication; Gene Expression Regulation, Plant; Genes, Plant; Genetic Variation; Genome, Plant; Malate Dehydrogenase (NADP+); Metabolic Networks and Pathways; Multigene Family; Phosphoenolpyruvate Carboxylase; Photosynthesis; Polymorphism, Single Nucleotide; Pyruvate, Orthophosphate Dikinase; Sorghum
PubMed: 32708598
DOI: 10.3390/genes11070806 -
Cell Death & Disease Aug 2022On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we...
On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.
Topics: Breast Neoplasms; Entosis; Female; Glucose; Glycosylation; Humans; Phosphoenolpyruvate Carboxykinase (ATP)
PubMed: 36002449
DOI: 10.1038/s41419-022-05177-x