High-Resolution Crystal Structure of Muscle Phosphoglycerate Mutase Provides Insight into Its Nuclear Import and Role.

Phosphoglycerate mutase (PGAM) is a glycolytic enzyme converting 3-phosphoglycerate to 2-phosphoglycerate, which in mammalian cells is expressed in two isoforms: brain (PGAM1) and muscle (PGAM2). Recently, it was shown that besides its enzymatic function, PGAM2 can be imported to the cell nucleus where it co-localizes with the nucleoli. It was suggested that it functions there to stabilize the nucleolar structure, maintain mRNA expression, and assist in the assembly of new pre-ribosomal subunits. However, the precise mechanism by which the protein translocates to the nucleus is unknown. In this study, we present the first crystal structure of PGAM2, identify the residues involved in the nuclear localization of the protein and propose that PGAM contains a "quaternary nuclear localization sequence (NLS)", i.e., one that consists of residues from different protein chains. Additionally, we identify potential interaction partners for PGAM2 in the nucleoli and demonstrate that 14-3-3ζ/δ is indeed an interaction partner of PGAM2 in the nucleus. We also present evidence that the insulin/IGF1-PI3K-Akt-mTOR signaling pathway is responsible for the nuclear localization of PGAM2.

Cobalt Regulates Activation of Camk2α in Neurons by Influencing Fructose 1,6-bisphosphatase 2 Quaternary Structure and Subcellular Localization.

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD+ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co2+, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co2+, and we provide a line of evidence that Co2+ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca2+/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.

Absolute Proteome Analysis of Hippocampus, Cortex and Cerebellum in Aged and Young Mice Reveals Changes in Energy Metabolism.

Aging is associated with a general decline of cognitive functions, and it is widely accepted that this decline results from changes in the expression of proteins involved in regulation of synaptic plasticity. However, several lines of evidence have accumulated that suggest that the impaired function of the aged brain may be related to significant alterations in the energy metabolism. In the current study, we employed the label-free "Total protein approach" (TPA) method to focus on the similarities and differences in energy metabolism proteomes of young (1-month-old) and aged (22-month-old) murine brains. We quantified over 7000 proteins in each of the following three analyzed brain structures: the hippocampus, the cerebral cortex and the cerebellum. To the best of our knowledge, this is the most extensive quantitative proteomic description of energy metabolism pathways during the physiological aging of mice. The analysis demonstrates that aging does not significantly affect the abundance of total proteins in the studied brain structures, however, the levels of proteins constituting energy metabolism pathways differ significantly between young and aged mice.

Expression of Fbp2, a Newly Discovered Constituent of Memory Formation Mechanisms, Is Regulated by Astrocyte-Neuron Crosstalk.

Fbp2 (muscle isozyme of fructose 1,6-bisphosphatase) is a glyconeogenesis-regulating enzyme and a multifunctional protein indispensable for long-term potentiation (LTP) formation in the hippocampus. Here, we present evidence that expression of Fbp2 in murine hippocampal cell cultures is regulated by crosstalk between neurons and astrocytes. Co-culturing of the two cell types results in a decrease in Fbp2 expression in astrocytes, and its simultaneous increase in neurons, as compared to monocultures. These changes are regulated by paracrine signaling using extracellular vesicle (EV)-packed factors released to the culture medium. It is well accepted that astrocyte-neuron metabolic crosstalk plays a crucial role in shaping neuronal function, and recently we have suggested that Fbp2 is a hub linking neuronal signaling with redox and/or energetic state of brain during the formation of memory traces. Thus, our present results emphasize the importance of astrocyte-neuron crosstalk in the regulation of the cells' metabolism and synaptic plasticity, and bring us one step closer to a mechanistic understanding of the role of Fbp2 in these processes.

Aging-associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte-to-neuron lactate shuttle.

Lactate derived from astrocytic glycogen has been shown to support memory formation in hippocampi of young animals, inhibiting it in old animals. Here we show, using quantitative mass spectrometry-based proteomics, immunofluorescence, and qPCR that aging is associated with an increase of glycogen metabolism enzymes concentration and shift in their localization from astrocytes to neurons. These changes are accompanied with reorganization of hippocampal energy metabolism which is manifested by elevated capacity of aging neurons to oxidize glucose in glycolysis and mitochondria, and decreased ability for fatty acids utilization. Our observations suggest that astrocyte-to-neuron lactate shuttle may operate in young hippocampi, however, during aging neurons become independent on astrocytic lactate and the metabolic crosstalk between the brain's cells is disrupted.

Effects of mutations in Wnt/ß-catenin, hedgehog, Notch and PI3K pathways on GSK-3 activity-Diverse effects on cell growth, metabolism and cancer.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that participates in an array of critical cellular processes. GSK-3 was first characterized as an enzyme that phosphorylated and inactivated glycogen synthase. However, subsequent studies have revealed that this moon-lighting protein is involved in numerous signaling pathways that regulate not only metabolism but also have roles in: apoptosis, cell cycle progression, cell renewal, differentiation, embryogenesis, migration, regulation of gene transcription, stem cell biology and survival. In this review, we will discuss the roles that GSK-3 plays in various diseases as well as how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, Wnt/beta-catenin, hedgehog, Notch and TP53. Mutations that occur in these and other pathways can alter the effects that natural GSK-3 activity has on regulating these signaling circuits that can lead to cancer as well as other diseases. The novel roles that microRNAs play in regulation of the effects of GSK-3 will also be evaluated. Targeting GSK-3 and these other pathways may improve therapy and overcome therapeutic resistance.

Proteomics Unveils Fibroblast-Cardiomyocyte Lactate Shuttle and Hexokinase Paradox in Mouse Muscles.

Quantitative mapping, given in biochemically interpretable units such as mol per mg of total protein, of tissue-specific proteomes is prerequisite for the analysis of any process in cells. We applied label- and standard-free proteomics to characterize three types of striated muscles: white, red, and cardiac muscle. The analysis presented here uncovers several unexpected and novel features of striated muscles. In addition to differences in protein expression levels, the three muscle types substantially differ in their patterns of basic metabolic pathways and isoforms of regulatory proteins. Importantly, some of the conclusions drawn on the basis of our results, such as the potential existence of a "fibroblast-cardiomyocyte lactate shuttle" and the "hexokinase paradox" point to the necessity of reinterpretation of some basic aspects of striated muscle metabolism. The data presented here constitute a powerful database and a resource for future studies of muscle physiology and for the design of pharmaceutics for the treatment of muscular disorders.

Absolute protein quantification allows differentiation of cell-specific metabolic routes and functions.

Total protein approach (TPA) is a proteomic method that allows calculation of concentrations of individual proteins and groups of functionally related proteins in any protein mixture without spike-in standards. Using the two-step digestion-filter-aided sample preparation method and LC-MS/MS analysis, we generated comprehensive quantitative datasets of mouse intestinal mucosa, liver, red muscle fibers, brain, and of human plasma, erythrocytes, and tumor cells lines. We show that the TPA-based quantitative data reflect well-defined and specific physiological functions of different organs and cells, for example nutrient absorption and transport in intestine, amino acid catabolism and bile secretion in liver, and contraction of muscle fibers. Focusing on key metabolic processes, we compared metabolic capacities in different tissues and cells. In addition, we demonstrate quantitative differences in the mitochondrial proteomes. Providing insight into the abundances of mitochondrial metabolite transporters, we demonstrate that their titers are well tuned to cell-specific metabolic requirements. This study provides for the first time a comprehensive overview of the protein hardware mediating metabolism in different mammalian organs and cells. The presented approach can be applied to any other system to study biological processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001352 (http://proteomecentral.proteomexchange.org/dataset/PXD001352).

Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.

Absolute quantitative profiling of the key metabolic pathways in slow and fast skeletal muscle.

Slow and fast skeletal muscles are composed of, respectively, mainly oxidative and glycolytic muscle fibers, which are the basic cellular motor units of the motility apparatus. They largely differ in excitability, contraction mechanism, and metabolism. Because of their pivotal role in body motion and homeostasis, the skeletal muscles have been extensively studied using biochemical and molecular biology approaches. Here we describe a simple analytical and computational approach to estimate titers of enzymes of basic metabolic pathways and proteins of the contractile machinery in the skeletal muscles. Proteomic analysis of mouse slow and fast muscles allowed estimation of the titers of enzymes involved in the carbohydrate, lipid, and energy metabolism. Notably, we observed that differences observed between the two muscle types occur simultaneously for all proteins involved in a specific process such as glycolysis, free fatty acid catabolism, Krebs cycle, or oxidative phosphorylation. These differences are in a good agreement with the well-established biochemical picture of the muscle types. We show a correlation between maximal activity and the enzyme titer, suggesting that change in enzyme concentration is a good proxy for its catalytic potential in vivo. As a consequence, proteomic profiling of enzyme titers can be used to monitor metabolic changes in cells. Additionally, quantitative data of structural proteins allowed studying muscle type specific cell architecture and its remodeling. The presented proteomic approach can be applied to study metabolism in any other tissue or cell line.

Absolute Proteome Analysis of Colorectal Mucosa, Adenoma, and Cancer Reveals Drastic Changes in Fatty Acid Metabolism and Plasma Membrane Transporters.

Colorectal cancer is a leading cause of cancer-related death. It develops from normal enterocytes, through a benign adenoma stage, into the cancer and finally into the metastatic form. We previously compared the proteomes of normal colorectal enterocytes, cancer and nodal metastasis to a depth of 8100 proteins and found extensive quantitative remodeling between normal and cancer tissues but not cancer and metastasis (Wisniewski et al. PMID 22968445). Here we utilize advances in the proteomic workflow to perform an in depth analysis of the normal tissue (N), the adenoma (A), and the cancer (C). Absolute proteomics of 10 000 proteins per patient from microdissected formalin-fixed and paraffin-embedded clinical material established a quantitative protein repository of the disease. Between N and A, 23% of all proteins changed significantly, 17.8% from A to C and 21.6% from N to C. Together with principal component analysis of the patient groups, this suggests that N, A, and C are equidistant but not on one developmental line. Our proteomics approach allowed us to assess changes in varied cell size, the composition of different subcellular components, and alterations in basic biological processes including the energy metabolism, plasma membrane transport, DNA replication, and transcription. This revealed several-fold higher concentrations of enzymes in fatty acid metabolism in C compared with N, and unexpectedly, the same held true of plasma membrane transporters.

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes.

Nuclear localization of aldolase A correlates with cell proliferation.

Muscle fructose 1,6-bisphosphate aldolase (ALDA) is a glycolytic enzyme which may localize both in nuclei and cytoplasm of cells, however its role in the nuclei is unclear. Here, we demonstrate the links between subcellular localization of ALDA and the cell cycle progression as well as the availability of energetic substrates. Results of our studies indicate that nuclear localization of ALDA correlates with the proliferative activity of the cells and with the expression of Ki-67, a marker of proliferation, both in the KLN-205 (mouse lung cancer cells) and human squamous cell lung cancer cells (hSCC). Chemically-induced block of cell cycle entry in S phase and the inhibition of transcription stimulate removal of ALDA from cells nuclei suggesting that nuclear ALDA is involved in cells proliferation. On the other hand, subcellular distribution of the enzyme also depends on the stress and pro-survival signals mediated by the Akt and the p38 pathways and, in non-proliferating cells, on the availability of glucose and lactate. The results presented here point to ALDA as a factor involved in the regulation of cells proliferation.

Destabilization of fructose 1,6-bisphosphatase-Z-line interactions is a mechanism of glyconeogenesis down-regulation in vivo.

Although it is well known that insulin controls the synthesis of glycogen from non-carbohydrates by down-regulating expression of several glyconeogenic enzymes, a mechanism of short-term inhibition of glyconeogenesis remains unknown. In recent years, we have shown that in skeletal muscle, fructose 1,6-bisphosphatase (FBPase) is a part of the hypothetical glyconeogenic complex located on sarcomeric Z-line. Here, we show that inhibition of glycogen synthase kinase-3 causes disruption of the FBPase-Z-line interactions and reduction of muscle glycogen content in vivo. The normal, striated pattern of muscle FBPase localization is also disturbed by insulin treatment but preserved when insulin is applied together with Akt inhibitor. We suggest that destabilization of FBPase-Z-line interaction is a universal cellular mechanism of glyconeogenesis down-regulation, allowing for preferential utilization of glucose for insulin-stimulated muscle glycogen synthesis.

Glycogen phosphorylase inhibition improves cognitive function of aged mice.

Inhibition of glycogen breakdown blocks memory formation in young animals, but it stimulates the maintenance of the long-term potentiation, a cellular mechanism of memory formation, in hippocampal slices of old animals. Here, we report that a 2-week treatment with glycogen phosphorylase inhibitor BAY U6751 alleviated memory deficits and stimulated neuroplasticity in old mice. Using the 2-Novel Object Recognition and Novel Object Location tests, we discovered that the prolonged intraperitoneal administration of BAY U6751 improved memory formation in old mice. This was accompanied by changes in morphology of dendritic spines in hippocampal neurons, and by "rejuvenation" of hippocampal proteome. In contrast, in young animals, inhibition of glycogen degradation impaired memory formation; however, as in old mice, it did not alter significantly the morphology and density of cortical dendritic spines. Our findings provide evidence that prolonged inhibition of glycogen phosphorolysis improves memory formation of old animals. This could lead to the development of new strategies for treatment of age-related memory deficits.

Association of C-terminal region of phosphoglycerate mutase with glycolytic complex regulates energy production in cancer cells.

Cancer cells prefer anaerobic ATP synthesis, regardless of the availability of oxygen. It has been hypothesized that in these cells, glycolytic enzymes associate into a large complex, which results in an increased efficiency of glycolytic flux. However, there is no convincing in vivo evidence supporting this hypothesis. Here, we show that all the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, form a macromolecular complex in vivo and that disruption of such complex significantly inhibits lactate release and ATP synthesis in the glycolytic pathway. Composition of the complex and the effectiveness of the glycolytic flux depends on lactate and glucose concentration. High concentrations of exogenous lactate reduces association of the C-terminal region phosphoglycerate mutase (PGAM) with the complex which results in its disruption and inhibition of ATP synthesis. Additionally, high lactate affects nuclear localization of PGAM and ceases cell proliferation. Our findings might provide new prospects for cancer treatment using low-molecular weight competitors to destabilize the glycolytic complex and reduce proliferative potential of cancer cells.

Cell cycle-dependent expression and subcellular localization of fructose 1,6-bisphosphatase.

Recently a gluconeogenic enzyme was discovered-fructose 1,6-bisphosphatase (FBPase)-that localizes in the nucleus of a proliferating cell, but its physiological role in this compartment remains unclear. Here, we demonstrate the link between nuclear localization of FBPase and the cell cycle progression. Results of our studies indicate that in human and mouse squamous cell lung cancer, as well as in the HL-1 cardiomyocytes, FBPase nuclear localization correlates with nuclear localization of S and G2 phase cyclins. Additionally, activity and expression of the enzyme depends on cell cycle stages. Identification of FBPase interacting partners with mass spectrometry reveals a set of nuclear proteins involved in cell cycle regulation, mRNA processing and in stabilization of genomic DNA structure. To our knowledge, this is the first experimental evidence that muscle FBPase is involved in cell cycle events.

FBP2-A New Player in Regulation of Motility of Mitochondria and Stability of Microtubules in Cardiomyocytes.

Recently, we have shown that the physiological roles of a multifunctional protein fructose 1,6-bisphosphatase 2 (FBP2, also called muscle FBP) depend on the oligomeric state of the protein. Here, we present several lines of evidence that in HL-1 cardiomyocytes, a forced, chemically induced reduction in the FBP2 dimer-tetramer ratio that imitates AMP and NAD+ action and restricts FBP2-mitochondria interaction, results in an increase in Tau phosphorylation, augmentation of FBP2-Tau and FBP2-MAP1B interactions, disturbance of tubulin network, marked reduction in the speed of mitochondrial trafficking and increase in mitophagy. These results not only highlight the significance of oligomerization for the regulation of FBP2 physiological role in the cell, but they also demonstrate a novel, important cellular function of this multitasking protein-a function that might be crucial for processes that take place during physiological and pathological cardiac remodeling, and during the onset of diseases which are rooted in the destabilization of MT and/or mitochondrial network dynamics.

Effects of the Mutant TP53 Reactivator APR-246 on Therapeutic Sensitivity of Pancreatic Cancer Cells in the Presence and Absence of WT-TP53.

The TP53 tumor suppressor is mutated in ~75% of pancreatic cancers. The mutant TP53 protein in pancreatic ductal adenocarcinomas (PDAC) promotes tumor growth and metastasis. Attempts have been made to develop molecules that restore at least some of the properties of wild-type (WT) TP53. APR-246 is one such molecule, and it is referred to as a mutant TP53 reactivator. To understand the potential of APR-246 to sensitize PDAC cells to chemotherapy, we introduced a vector encoding WT-TP53 into two PDAC cell lines, one lacking the expression of TP53 (PANC-28) and one with a gain-of-function (GOF) mutant TP53 (MIA-PaCa-2). APR-246 increased drug sensitivity in the cells containing either a WT or mutant TP53 protein with GOF activity, but not in cells that lacked TP53. The introduction of WT-T53 into PANC-28 cells increased their sensitivity to the TP53 reactivator, chemotherapeutic drugs, and signal transduction inhibitors. The addition of WT-TP53 to PDAC cells with GOF TP53 also increased their sensitivity to the drugs and therapeutics, indicating that APR-246 could function in cells with WT-TP53 and GOF TP53. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function through the reactivation of TP53.

Structural studies of human muscle FBPase.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).