Antrodia cinnamomea Inhibits Migration in Human Hepatocellular Carcinoma Cells: The Role of ERp57 and PGK-1.

Evidences suggest that ERp57 and PGK-1 signaling lead to cancer cell proliferation and migration. We hypothesized that ERp57 and PGK-1 down-regulation may inactivate matrix metalloproteinase (MMP)-2, -9 expressions and inhibit hepatocellular carcinoma (HCC) migration. Antrodia cinnamomea is widely prescribed as an adjuvant to treat HCC in Taiwan. We aimed to investigate if ethanol extract of fruiting bodies of Antrodia cinnamomea (EEAC) and its active ingredients (i.e., zhankuic acid A, cordycepin, and adenosine) can modulate HCC cancer cells migration through ERp57 and PGK-1 and other molecular pathways such as PI3K/Akt and MAPK. ERp57 and PGK-1 siRNA were transfected into HCC to determine effects on MMP-2/-9 expressions and cell migration. We then examined the inhibitory effects of EEAC and its active ingredients on HCC migration and its related mechanisms including ERp57, PGK-1, PI3K/Akt, and MAPK signaling pathways. Down-regulation of ERp57 and PGK-1 by siRNA decreased MMP-2, -9 expressions and Transwell cell migration in HCC. Nontoxic EEAC markedly inhibited migration of HCC, and significantly inhibited activities and protein expressions of MMP-2 and -9, while the expression of the endogenous inhibitors (TIMP-1 and TIMP-2) of these proteins increased. Nontoxic EEAC and its active ingredients decreased ERp57, GLUD-1, GST-pi, and PGK-1 protein expressions. Finally, nontoxic EEAC inhibited the phosphorylated FAK, PI3K/Akt, and MAPK signaling. Our findings first indicate that EEAC and its ingredients effectively suppress HCC migration. Additionally, the molecular mechanisms appear to be mediated, in part, through the down-regulation of ERp57, PGK-1, MAPK, and PI3K/Akt.

The phosphoglycerate kinase isoenzymes have distinct roles in the regulation of carbohydrate metabolism in Trypanosoma cruzi.

The glycolytic enzyme phosphoglycerate kinase (PGK) is present in Trypanosoma cruzi as three isoenzymes, two of them located inside glycosomes (PGKA and PGKC) and another one in the cytosol (PGKB). The three isoenzymes are expressed at all stages of the life cycle of the parasite. A heterologous expression system for PGKA (rPGKA) was developed and the substrate affinities of the natural and recombinant PGKA isoenzyme were determined. Km values measured for 3-phosphoglycerate (3PGA) were 174 and 850 µM, and for ATP 217 and 236 µM, for the natural and recombinant enzyme, respectively. No significant differences were found between the two forms of the enzyme. The rPGKA was inhibited by Suramin with Ki values of 10.08 µM and 12.11 µM for ATP and 3PGA, respectively, and the natural enzyme was inhibited at similar values. A site-directed mutant was created in which the 80 amino acids PGKA sequence, present as a distinctive insertion in the N-terminal domain, was deleted. This internally truncated PGKA showed the same Km values and specific activity as the full-length rPGKA. The natural PGKC isoenzyme was purified from epimastigotes and separated from PGKA through molecular exclusion chromatography and its kinetic characteristics were determined. The Km value obtained for 3PGA was 192 µM, and 10 µM for ATP. Contrary to PGKA, the activity of PGKC is tightly regulated by ATP (substrate inhibition) with a Ki of 270 µM, suggesting a role for this isoenzyme in regulating metabolic fluxes inside the glycosomes.

GAPDH binds to active Akt, leading to Bcl-xL increase and escape from caspase-independent cell death.

Increased glucose catabolism and resistance to cell death are hallmarks of cancers, but the link between them remains elusive. Remarkably, under conditions where caspases are inhibited, the process of cell death is delayed but rarely blocked, leading to the occurrence of caspase-independent cell death (CICD). Escape from CICD is particularly relevant in the context of cancer as apoptosis inhibition only is often not sufficient to allow oncogenic transformation. While most glycolytic enzymes are overexpressed in tumors, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of particular interest as it can allow cells to recover from CICD. Here, we show that GAPDH, but no other glycolytic enzymes tested, when overexpressed could bind to active Akt and limit its dephosphorylation. Active Akt prevents FoxO nuclear localization, which precludes Bcl-6 expression and leads to Bcl-xL overexpression. The GAPDH-dependent Bcl-xL overexpression is able to protect a subset of mitochondria from permeabilization that are required for cellular survival from CICD. Thus, our work suggests that GAPDH overexpression could induce Bcl-xL overexpression and protect cells from CICD-induced chemotherapy through preservation of intact mitochondria that may facilitate tumor survival and chemotherapeutic resistance.

Involvement of a minimal actin-binding region of Spiroplasma citri phosphoglycerate kinase in spiroplasma transmission by its leafhopper vector.

BACKGROUND: Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49-154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His(6)-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission. CONCLUSION: These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.

Physiology and endocrinology symposium: a proteome-based model for sperm mobility phenotype.

Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca(2+) overloading. The hypothesis was rejected because staining with a cell permeant Ca(2+)-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks.

Recovery of functional enzyme from amyloid fibrils.

Amyloid deposits, which accumulate in numerous diseases, are the final stage of multi-step protein conformational-conversion and oligomerization processes. The underlying molecular mechanisms are not fully understood, and particularly little is known about the reverse reaction. Here we show that phosphoglycerate kinase amyloid fibrils can be converted back into native protein. We achieved recovery with 60% efficiency, which is comparable to the success rate of the unfolding-refolding studies, and the recovered enzyme was folded, stable and fully active. The key intermediate stages in the recovery process are fibril disassembly and unfolding followed by spontaneous protein folding.

Polypyrimidine tract binding protein 2 stabilizes phosphoglycerate kinase 2 mRNA in murine male germ cells by binding to its 3'UTR.

The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3'-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3'-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3'-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.

Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus.

The tertiary structure in the 3'-untranslated region (3'-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3'-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3'-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3'-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced.

[Magnesium magnetic isotope effect: a key towards mechanochemistry of phosphorylating enzymes as molecular machines].

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.

GAPDH enhances group II intron splicing in vitro.

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.

Regulation of urokinase receptor expression by phosphoglycerate kinase.

Post-transcriptional regulation represents a major mechanism by which eukaryotic gene expression is regulated through cis-trans interactions that serve as signals for rapid alterations of messenger RNA (mRNA) stability. Regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with a 50 kD uPAR mRNA binding protein. We purified this protein from human bronchial epithelial (Beas2B) cells and identified it as phosphoglycerate kinase (PGK). We cloned PGK cDNA by polymerase chain reaction and expressed the recombinant PGK protein, which specifically bound the uPAR mRNA coding region by gel mobility shift and Northwestern blotting. We also confirmed a direct interaction of PGK protein with uPAR mRNA by immunoprecipitation. Overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells resulted in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression. Reduced uPAR mRNA expression involved decreased stability of the uPAR mRNA. Decline in 3H-thymidine incorporation and migration occurred in H157 cells transfected with PGK cDNA. These results demonstrate that PGK regulates uPAR expression at the post-transcriptional level.

Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents.

l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.

Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies.

We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.

Identification of annexin II heterotetramer as a plasmin reductase.

Annexin II heterotetramer (AIIt) is a Ca(2+)- and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A(61) (Lys(78)-Lys(468)) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys(462)-Cys(541) bond in a time- and concentration-dependent manner, which resulted in the release of A(61) from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A(61) from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A(61).

Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide.

Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.

Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea pig cardiac myocytes.

In heart, glycolysis may be a preferential source of adenosine triphosphate (ATP) for membrane functions. In this study the patch-clamp technique was used to study potassium channels sensitive to intracellular ATP levels in permeabilized ventricular myocytes. Activation of these K+ channels has been implicated in marked cellular K+ loss leading to electrophysiological abnormalities and arrhythmias during myocardial ischemia. The results showed that glycolysis was more effective than oxidative phosphorylation in preventing ATP-sensitive K+ channels from opening. Experiments in excised inside-out patches suggested that key glycolytic enzymes located in the membrane or adjacent cytoskeleton near the channels may account for their preference for glycolytic ATP.

Evolutionary conservation of the substrate-binding cleft of phosphoglycerate kinases.

The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve beta-sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3-phosphoglycerate-binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner-loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner-loop region. Trypanosome PGK contains a polypeptide segment in its N-terminal domain that is transposed in comparison with the other species.

Reproductive implications of LDH-C4 and other testis-specific isozymes.

This paper briefly reviews the biological properties and occurrence of selected testis-specific isozymes. These include phosphoglycerate kinase B, cytochrome ct, enolase S, and lactate dehydrogenase C4 (LDH-C4). The most extensively studies of these, LDH-C4, is described in detail with particular reference to its potential in an immunocontraceptive technology.

Energy metabolism in human erythrocytes: the role of phosphoglycerate kinase in cation transport.

Three models of disturbed erythrocyte metabolism, triose-depleted normal, phosphoglycerate kinase (PGK)-deficient, and pyruvate kinase (PK)-deficient cells, have been studied to examine further the role of PGK in erythrocyte cation transport. Sodium (Na-+) and potassium (K-+) transport were reduced only in cells fully depleted of triose. In such cells the PGK step presumably was inoperative due to total lack of substrate; 2,3-diphosphoglycerate (2,3-DPG) then became the sole substrate source for remaining steps in glycolysis. At increased intracellular Na-+ concentrations which normally stimulate transport and glycolysis, triose-depleted cells had marked impairment of cation transport and ouabain-inhibitable lactate and pyruvate production from 2,3-DPG. PGK-deficient cells and normal cells with high intracellular Na-+ concentrations had similar increases in transport and ouabain-inhibitable lactate production. PK-deficient cells with high intracellular Na-+ concentrations showed an appropriate increase in transport but less stimulation of lactate production. Transport was not related to total cellular adenosine triphosphate (ATP) concentration. These data suggested that normal coupled cation transport occurred despite diminished metabolite flow through PGK, as in PGK- or PK-deficient cells. Transport was diminished only in triose-depleted cells where metabolite flow through PGK was presumably absent. These data, therefore, support the concept that transport and glycolysis interact at the PGK step, although impairment of PGK must be profound before its effect on transport is evident.