Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.     

Regulation of nuclear phospholipase C activity

A body of evidence, linking inositide-specific phospholipase C (PI-PLC) to the nucleus, is quite extensive. The main isoform in the nucleus is PI-PLCbeta1, whose activity is up-regulated in response to insulin-like growth factor-1 (IGF-1) or insulin stimulation. Whilst at the plasma membrane this PI-PLC is activated and regulated by Galphaq/alpha(11) and Gbetagamma subunits, there is yet no evidence that qalpha/alpha(11) is present within the nuclear compartment, neither GTP-gamma-S nor AlF4 can stimulate PI-PLCbeta1 activity in isolated nuclei. Here we review the evidence that upon occupancy of type 1 IGF receptor there is translocation to the nucleus of phosphorylated mitogen-activated protein kinase (MAPK) which phosphorylates nuclear PI-PLCbeta1 and triggers its signalling, hinting at a separate pathway of regulation depending on the subcellular location of PI-PLCbeta1. The difference in the regulation of the activity of PI-PLCbeta1mirrors the evidence that nuclear and cytoplasmatic inositides can differ markedly in their signalling capability. Indeed, we do know that agonists which affect nuclear inositol lipid cycle at the nucleus do not stimulate the one at the plasma membrane.     

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.     

Identification and characterization of a phosphoinositide phosphate kinase homolog

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).     

Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements

Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.     

P311 binds to the latency associated protein and downregulates the expression of TGF-beta1 and TGF-beta2

P311 is an 8-kDa protein originally found in neurons and muscle. We recently showed that expression of P311 in NIH 3T3 cells induced a myofibroblast phenotype with low TGF-beta1 expression. Here we demonstrate that P311 downregulates not only TGF-beta1, but also TGF-beta2, expression, with no effect on TGF-beta3. In addition, P311 interacts with TGF-beta2 in a yeast two-hybrid system through a sequence encompassing part of the TGF-beta latent associated protein (LAP) and part of mature TGF-beta2. Coimmunoprecipitations demonstrated interaction between P311 and TGF-beta1 and 2, but not TGF-beta3. Additional coimmunoprecipitations after introducing LAP or mature TGF-beta1 into cells demonstrated P311 binding to LAP, but not to mature TGF-beta. P311 has a conserved PEST domain, which generally serves as a rapid degradation signal. Deletion of the PEST domain reversed the effect of P311 on TGF-beta isoforms. Finally, Smad3 activity was decreased in P311-expressing cells, but was corrected by exogenous TGF-beta1 treatment, which also elevated TGF-beta1 mRNA level. This suggested that P311 downregulates TGF-beta1 and 2 in part by blocking TGF-beta autoinduction.     

A novel fold revealed by Mycobacterium tuberculosis NAD kinase, a key allosteric enzyme in NADP biosynthesis

NAD kinase catalyzes the magnesium-dependent phosphorylation of NAD, representing the sole source of freshly synthesized NADP in all organisms. The enzyme is essential for the growth of the deadly multidrug-resistant pathogen Mycobacterium tuberculosis and is an attractive target for novel antitubercular agents. The crystal structure of NAD kinase has been solved by multiwavelength anomalous dispersion at a resolution of 2.3 A in its T state. Two crystal forms have been obtained revealing either a dimer or a tetramer. The enzyme architecture discloses a novel molecular arrangement, with each subunit consisting of an alpha/beta N-terminal domain and a C-terminal 12-stranded beta sandwich domain, connected by swapped beta strands. The C-terminal domain shows a striking internal approximate 222 symmetry and an unprecedented topology, revealing a novel fold within the family of all beta structures. The catalytic site is located in the long crevice that defines the interface between the domains. The conserved GGDG structural fingerprint of the catalytic site is reminiscent of the related region in 6-phosphofructokinase, supporting the hypothesis that NAD kinase belongs to a newly reported superfamily of kinases.     

Trypanosoma cruzi: mixture of two populations can modify virulence and tissue tropism in rat

In rats, CL-Brener clone caused high mortality, severe acute myocarditis, and myositis that subsided completely in surviving animals. Accordingly, no parasite kDNA could be amplified in several organs after 4 months. The monoclonal JG strain caused null mortality, acute predominantly focal myocarditis, discrete and focal myositis, and a chronic phase with sparse inflammatory foci. Double infection with both Trypanosoma cruzi populations turned mortality very low or null. At the end of the acute phase, the heart exhibited only JG strain kDNA (LSSP-PCR), while skeletal muscles and rectum exhibited only CL-Brener kDNA. Molecular and histopathological findings were accordant. In double infection chronic phase, JG strain remains in heart and appeared in organs previously parasitized by CL-Brener clone. Understanding the virulence and histotropism shifts now described could be important to clarify the variable clinical course and epidemiological peculiarities of Chagas' disease.