A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression.     

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism

Background

Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved.

Methodology/Principal Findings

In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux.

Conclusions/Significance

Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.     

Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.     

Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis,leads to metabolic impairment and autophagy induction in retinal pigment epithelium

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.     

Effects of the Yeast RNA-Binding Protein Whi3 on the Half-Life and Abundance of CLN3 mRNA and Other Targets

Whi3 is an RNA binding protein known to bind the mRNA of the yeast G1 cyclin gene CLN3. It inhibits CLN3 function, but the mechanism of this inhibition is unclear; in previous studies, Whi3 made no observable difference to CLN3 mRNA levels, translation, or protein abundance. Here, we re-approach this issue using microarrays, RNA-Seq, ribosome profiling, and other methods. By multiple methods, we find that the whi3 mutation causes a small but consistent increase in the abundance of hundreds of mRNAs, including the CLN3 mRNA. The effect on various mRNAs is roughly in proportion to the density of GCAU or UGCAU motifs carried by these mRNAs, which may be a binding site for Whi3. mRNA instability of Whi3 targets may in part depend on a 3′ AU rich element (ARE), AUUUUA. In addition, the whi3 mutation causes a small increase in the translational efficiency of CLN3 mRNA. The increase in CLN3 mRNA half-life and abundance together with the increase in translational efficiency is fully sufficient to explain the small-cell phenotype of whi3 mutants. Under stress conditions, Whi3 becomes a component of P-bodies or stress granules, but Whi3 also acts under non-stress condition, when no P-bodies are visible. We suggest that Whi3 may be a very broadly-acting, but mild, modulator of mRNA stability. In CLN3, Whi3 may bind to the 3′ GCAU motifs to attract the Ccr4-Not complex to promote RNA deadenylation and turnover, and Whi3 may bind to the 5′ GCAU motifs to inhibit translation.     

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

The G₁ cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.     

Mutations in <Emphasis Type="Italic">CDC14</Emphasis> result in high sensitivity to cyclin gene dosage in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14.     

Genetic and physiological evidence for the existence of a second glycolytic pathway in yeast parallel to the phosphofructokinase-aldolase reaction sequence

Summary Yeast mutants lacking phosphofructokinase activity because of a defect in one of the two genes PFK1 and PFK2 can still perform glycolysis and produce ethanol. However, they differ from normal wild-type yeast in several ways. After a transfer from a sugar-free to a glucose medium, wild-type cells start to produce ethanol right away, mutants only after a lag period of about 90 min. About two-thirds of the carbon atoms released as CO₂ from wild-type cells derive from glucose carbon atoms 3 and 4. Mutants with a single defect in one of the two phosphofructokinase genes PFK1 and PFK2 show no such a preferential contribution of these two C-atoms of glucose. All six C-atoms contribute almost equally to CO₂ production. We have isolated mutants that block glycolysis in single pfk1 and pfk2 mutants. They could be located in three different genes called BYP1, BYP2 and BYP3 (BYP for bypass). In a byp1 mutant, CO₂ derived almost exclusively from C-atoms 3 and 4 of glucose. This is what the classical concept of yeast glycolysis predicts. During a search for metabolites accumulating in pfk and byp mutants, we found sedoheptulose-7-phosphate, a pentosephosphate cycle intermediate not detectable in wild-type cells. An analysis of enzymes acting in the direct oxidation of glucose-6-phosphate and in the pentosephosphate cycle did not show any defects in those activities. It is hypothesized that the pentosephosphate cycle not only functions, in providing phosphorylated derivatives of tetroses and pentoses for biosynthetic needs, but also plays an important role in sugar catabolism and fermentation. This hypothesis also implies that the reaction sequency catalyzed by phosphofructokinase and aldolase covers only part of the total catabolic flux.     

Plant-like phosphofructokinase from Plasmodium falciparum belongs to a novel class of ATP-dependent enzymes

Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK β and α subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene β and α regions were cloned and expressed as His₆- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9β possessed catalytic activity. Monoclonal antibodies against the recombinant β protein confirmed that the PfPFK9 protein has β and α domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans.     

Cloning of a second gene encoding 6-phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate

We have identified a new gene, PFK27, that encodes a second inducible 6-phosphofructo-2-kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose-2,6-bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild-type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose-2,6-bisphosphate is an important effector in vivo of the 6-phosphofructo-1-kinase/fructose-1,6-bisphospha-tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

Effect of the β-amyloid peptide Aβ<Subscript>25–35</Subscript> and fullerene C<Subscript>60</Subscript> on the activity of enzymes in erythrocytes

The effect of the β-amyloid peptide Aβ_25–35 and fullerene C₆₀ on the activity of the cytoplasmic enzymes lactate dehydrogenase (LDH) and glutathione peroxidase (GLP), and membrane-bound phosphofructokinase (PFK) and Na⁺,K⁺-ATPase in human erythrocytes has been studied. When used in combination, the cytotoxins decrease the activity of LDH and PFK in a nonadditive manner; in this case, Aβ_25–35 protects PFK against the inhibitory effect of C₆₀. The activity of LDH, GLP, and PFK decreases within the first 2–20 min of incubation of erythrocytes with Aβ_25–35 in the absence of glucose. The addition of glucose sharply decreases the inhibitory action of Aβ_25–35 on LDH and GLP but does not affect the fourfold decrease in activity of PFK; the activity of membrane-bound Na⁺,K⁺-ATPase does not depend on the presence of glucose. Possible mechanisms of interaction of Aβ_25–35 and fullerene C₆₀ with the erythrocyte membrane and enzymes are discussed.     

Identification of Cdc45p,an essential factor required for DNA replication

CDC45 is an essential gene required for initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. CDC45 interacts genetically with CDC46 and CDC47, both members of the MCM family of genes which have been implicated in the licensing of DNA replication. In this report, the isolation of CDC45 is described. The complementing gene is linked to an essential open reading frame on chromosome XII. CDC45 was found to be cell cycle regulated and steady-state mRNA levels are G1/S-specific. CDC45 encodes a protein structurally related to Tsd2p, a protein required for DNA replication in Ustilago maydis. CDC45 also interacts genetically with ORC2, the gene encoding the second subunit of the origin recognition complex, ORC, and MCM3, another member of the MCM family. The cdc45-1 mutant has a plasmid maintenance defect which is rescued by the addition of multiple potential origins to the plasmid.