Andrzej Dzugaj

Kinetic properties of pig (Sus scrofa domestica) and bovine (Bos taurus) D-fructose-1,6-bisphosphate 1-phosphohydrolase (F1,6BPase): liver-like isozymes in mammalian lung tissue.

F1,6BPases from porcine and bovine lung were isolated and their kinetic properties were determined. Ks, Kis and beta were determined assuming partial-noncompetitive inhibition (simple intersecting hyperbolic noncompetitive inhibition) of the enzyme by the substrate. Values for Ks were 4.1 and 4.4 microM for porcine and bovine F1,6BPase, respectively and values for 1 were close to 0.55 in both cases. Kis were 9 and 15 microM for porcine and bovine F1,6BPase, respectively. I0.5 for AMP were determined as 7 microM for pig enzyme and 14 microM for F1,6BPase from bovine lung. The enzymes were inhibited by F2,6BP with Kis of 0.19 and 0.21 microM for porcine and bovine enzymes, respectively. In the presence of AMP concentration equal to I0.5, the Ki values for pig and bovine enzymes were 0.07 and 0.09 microM, respectively. The levels of F2,6BP, AMP and antioxidant enzymes activities in pig and bovine lung tissues were also determined. The cDNA coding sequence of pig lung F1,6BPase1 showed a high homology with pig liver enzyme, differing only in four positions (G/C-63, T/A-808, G/C-884 and T/A-1005) resulting in a single amino acid substitution (Gly-295 for Ala-295). It is hypothesized that the lung F1,6BPase participates in gluconeogenesis, surfactant synthesis and antioxidant reactions.

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase–aldolase complex is located on α‐actinin of the Z‐line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase–aldolase and FBPase‐α‐actinin complex, causes fast dissociation of FBPase from the Z‐line and slow accumulation of aldolase within the I‐band and M‐line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.

Calcium inhibits muscle FBPase and affects its intracellular localization in cardiomyocytes.

As our recent investigation revealed, in mammalian heart muscle, fructose 1,6‐bisphosphatase (FBPase) – a key enzyme of glyconeogenesis – is located around the Z‐line, inside cells nuclei and, as we demonstrate here for the first time, it associates with intercalated discs. Since the degree of association of numerous enzymes with subcellular structures depends on the metabolic state of the cell, we studied the effect of elevated Ca2+ concentration on localization of FBPase in cardiomyocytes. In such conditions, FBPase dissociated from the Z‐line, but no visible effect on FBPase associated with intercalated discs or on the nuclear localization of the enzyme was observed. Additionally, Ca2+ appeared to be a strong inhibitor of muscle FBPase.

Alteration of Akt activity increases chemotherapeutic drug and hormonal resistance in breast cancer yet confers an achilles heel by sensitization to targeted therapy

The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance.

Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition

Real‐time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase–aldolase complex revealed apparent binding constant [K Aapp] values of about 4.4×108 M−1. The stability of the complex was down‐regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6‐phosphate, and by the regulator of glycolysis and glyconeogenesis – fructose 2,6‐bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I 0.5 was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase–aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates’ myocytes.

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

Abstract AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 uM and 4.8 uM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the Nterminal region of the muscle enzyme towards liver FBPase (Lys20→Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20→Lys and double mutation Glu19→Asp/Glu20→Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 uM). The decrease of sensitivity for AMP of the muscle mutant Lys20→Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20→Lys and Glu19→Asp/Glu20→Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity, and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.).

The effect of a high dose of cortisol (200 mg kg(-1) body mass) on juvenile carp was investigated. The activity of glucose-6-phosphatase in liver and of fructose-1,6-bisphosphatase in liver, kidney and muscle, the serum glucose and fructose-2,6-bisphosphate concentration as well as the serum concentration of the injected hormone were measured after 24, 72 and 216 h after intraperitoneal cortisol injection. The activities of fructose-1,6-bisphosphatase in liver and kidney and glucose-6-phosphatase in liver were elevated in comparison with the control, while the fructose-1,6-bisphosphatase activity in the muscle tissue was unchanged. After cortisol injection, the serum glucose level was nearly two times higher after 24 and 72 h and was still 50% higher after 216 h compared with controls. In contrast, the liver fructose-2,6-bisphosphate concentration was unchanged after 24 h. More than two times higher fructose-2,6-bisphosphate concentration was observed in liver after 72 h and it was still elevated after 216 h after the cortisol injection.

Evolutionary conserved N‐terminal region of human muscle fructose 1,6‐bisphosphatase regulates its activity and the interaction with aldolase

N‐terminal residues of muscle fructose 1,6‐bisphosphatase (FBPase) are highly conserved among vertebrates. In this article, we present evidence that the conservation is responsible for the unique properties of the muscle FBPase isozyme: high sensitivity to AMP and Ca2+ inhibition and the high affinity to muscle aldolase, which is a factor desensitizing muscle FBPase toward AMP and Ca2+. The first N‐terminal residue affecting the affinity of muscle FBPase to aldolase is arginine 3. On the other hand, the first residue significantly influencing the kinetics of muscle FBPase is proline 5. Truncation from 5–7 N‐terminal residues of the enzyme not only decreases its affinity to aldolase but also reduces its k‐cat and activation by Mg2+, and desensitizes FBPase to inhibition by AMP and calcium ions. Deletion of the first 10 amino acids of muscle FBPase abolishes cooperativity of Mg2+ activation and results in biphasic inhibition of the enzyme by AMP. Moreover, this truncation lowers affinity of muscle FBPase to aldolase about 14 times, making it resemble the liver isozyme. We suggest that the existence of highly AMP‐sensitive muscle‐like FBPase, activity of which is regulated by metabolite‐dependent interaction with aldolase enables the precise regulation of muscle energy expenditures and might contributed to the evolutionary success of vertebrates. Proteins 2008.

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions

Muscle fructose‐1,6‐bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52–72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu69/Gln were employed. The chemical modification of Lys71–72 and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu69 → Gln resulted in a 500‐fold increase of muscle isozyme I 0.5 vs. calcium ions. These results demonstrate the key role that the 52–72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.

Colocalization of aldolase and FBPase in cytoplasm and nucleus of cardiomyocytes

The protein exchange method, immunocytochemistry and the nuclear import of fluorophore‐labeled enzymes were used to investigate the colocalisation of aldolase and FBPase in cardiomyocytes. The results indicate in vivo interaction of these two enzymes. In the cardiomyocyte cytoplasm, these enzymes were found to colocalise at the Z‐line and on intercalated discs. The translocation of both enzymes through the nuclear pores was also investigated. The immunocytochemistry revealed the colocalisation of aldolase and FBPase in the heterochromatin region of cardiomyocyte nuclei. The Pearsons correlation coefficients, which represent the degree of colocalisation were 0.47, 0.52 and 0.66 in the sarcomer, the intercalated disc and the nucleus, respectively. This is the first report on aldolase and FBPase colocalisation in cardiomyocytes. Interaction of aldolase with FBPase, which results in heterologous complex formation, is necessary for glyconeogenesis to proceed. Therefore, this metabolic pathway in the sarcomer, in the intercalated disc as well as in the nucleus might be expected.