Volume 390, Issue 3

Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases of aging, cystic fibrosis, and cardiovascular, inflammatory, immune, metabolic, and neurodegenerative diseases. Owing to the pleiotropic effects of GSH on cell functions, it has been quite difficult to define the role of GSH in the onset and/or the expression of human diseases, although significant progress is being made. GSH levels, turnover rates, and/or oxidation state can be compromised by inherited or acquired defects in the enzymes, transporters, signaling molecules, or transcription factors that are involved in its homeostasis, or from exposure to reactive chemicals or metabolic intermediates. GSH deficiency or a decrease in the GSH/glutathione disulfide ratio manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in diseases, such as cancer, Parkinson's disease, and Alzheimer's disease. In addition, imbalances in GSH levels affect immune system function, and are thought to play a role in the aging process. Just as low intracellular GSH levels decrease cellular antioxidant capacity, elevated GSH levels generally increase antioxidant capacity and resistance to oxidative stress, and this is observed in many cancer cells. The higher GSH levels in some tumor cells are also typically associated with higher levels of GSH-related enzymes and transporters. Although neither the mechanism nor the implications of these changes are well defined, the high GSH content makes cancer cells chemoresistant, which is a major factor that limits drug treatment. The present report highlights and integrates the growing connections between imbalances in GSH homeostasis and a multitude of human diseases.

Porcine reproductive and respiratory syndrome (PRRS) virus is an RNA virus that replicates in the cytoplasm, but the viral nucleocapsid (N) protein localizes specifically in the nucleus and nucleolus of virus-infected cells. Nuclear localization of N is non-essential for PRRSV replication in cultured cells but has been shown to modulate the pathogenesis of virus in pigs, suggesting that N plays an accessory role in the nucleus during infection. We identified by yeast two-hybrid screening the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) as a cellular partner for PRRS virus (PRRSV) N protein. This protein is a homolog of human HIC, a recently identified cellular transcription factor. The specific interaction of PRRSV N with HIC was confirmed in cells by mammalian two-hybrid assay and co-immunoprecipitation and in vitro by GST pull-down assay. HIC is a zinc-binding protein and confocal microscopy demonstrated co-localization of N with the HIC-p40 isomer in the nucleus and nucleolus, and in the cytoplasm with HIC-p32, which is the N-terminal truncation of HIC-p40. The porcine homolog of HIC is universally expressed in pig tissues including alveolar macrophages. The interaction of viral capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection.

Human eosinophil-derived neurotoxin (EDN), a secretory protein from eosinophils, is a member of the RNase A superfamily. The ribonucleolytic activity of EDN is central to its biological activities. EDN binds RNA in a cationic cleft, and the interaction between EDN and RNA substrate extends beyond the scissile bond. Based on its homology with RNase A, putative substrate binding subsites have been identified in EDN. The B 1 and B 2 subsites interact specifically with bases, whereas P 0 , P 1 , and P 2 subsites interact with phosphoryl groups. In this study, we evaluated the role of putative residues of these subsites in the ribonucleolytic activity of EDN. We demonstrate that of the two base binding subsites, B 1 is critical for the catalytic activity of EDN, as the substrate cleavage was dramatically reduced upon substitution of B 1 subsite residues. Among the phosphate-binding subsites, P 1 is the most crucial as mutations of its constituting residues totally abolished the catalytic activity of EDN. Mutation of P 0 and P 2 subsite residues only affected the catalytic activity on the homopolymer Poly(U). Our study demonstrates that P 1 and B 1 subsites of EDN are critical for its catalytic activity and that the other phosphate-binding subsites are involved in the activity on long homopolymeric substrates.

Members of all three classes of the protein kinase C (PKC) family including atypical PKCzeta (PKCζ) are involved in central functions of liver parenchymal cells. However, expression and localization of PKCiota (PKCι), the highly homologous atypical PKC (aPKC) isoform, in hepatocytes is unknown to date. PKCζ and PKCι were cloned from human and rat liver and fused to fluorescent protein tags (YFP). The sequence of full-length rat PKCι is not yet known and was cloned from cDNA of hepatocytes by the use of degenerated primers. PKCζ-YFP and PKCι-YFP (human and rat) were expressed in HeLa or HEK293 cells and used to test the specificity of seven aPKC antibodies. Two antibodies were PKCι-specific and two were specific for PKCζ in immunofluorescence and Western blot analysis. Subcellular localization was analyzed by immunofluorescence in isolated rat and human hepatocytes and liver sections. Low immunoreactivity for aPKCs was found at the sinusoidal membrane and in the cytosol. The highest density of PKCι as well as PKCζ was found at the canalicular membrane in co-localization with ABC-transporters, such as bile salt export pump or multidrug resistance-associated protein 2. This topology suggests a specific function of aPKCs at the canalicular membrane in addition to their known role in cell polarity of epithelial cells.

The effect of viral infection on the regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in Nicotiana tabacum L. leaves was studied. PEPC activity was 3 times higher in infected plant leaves compared to healthy plants. Activity of plant PEPC can be regulated, e.g., by de novo synthesis or reversible phosphorylation. The reason for the increase of PEPC activity as a consequence of PVY NTN infection was studied. The amount of PEPC determined by Western blot analysis or by relative estimation of PEPC mRNA by real-time PCR did not differ in control and PVY NTN -infected plants. Changes in post-translational modification of PEPC by phosphorylation were evaluated by comparing activity of the native and the dephosphorylated enzyme. The infected plants were characterized by a higher decrease of the enzyme activity after its dephosphorylation, which indicated a higher phosphorylation level. Immunochemical detection of phosphoproteins by Western blot analysis showed a more intensive band corresponding to PEPC from the infected material. This strengthens the hypothesis of an infection-related phosphorylation, which could be part of the plant's response to pathogen attack. The physiological implications of the increase in PEPC activity during PVY NTN infection are discussed.

Phospholipase D (PLD) is a receptor-regulated signalling enzyme involved in biological functions, such as exocytosis, phagocytosis, actin dynamics, membrane trafficking, and is considered to be essential for stimulated degranulation of cells. The purpose of our investigation was to examine how the fatty acid pattern of cellular membranes influences the activities and cellular distribution of the PLD1 and PLD2 isoforms. Expression of GFP-tagged PLD1 and PLD2 in COS-1 cells that were stimulated with mastoparan after cultivation in 20 μmol linoleic (C18:2n6) or linolenic (C18:3n3) acid for 4 d demonstrated that PLD1 dramatically alters its cellular distribution and is redistributed from intracellular vesicles to the cell surface. PLD2, on the other hand, maintains its localisation at the plasma membrane. The activity of PLD, which corresponds to PLD1 and PLD2, significantly increased two- to three-fold in the presence of the fatty acids. We conclude that linoleic acid and linolenic acid supplementation affect the intracellular trafficking of the PLD1 isoform and the activity of PLD most likely due to alterations in the membrane lipid environment conferred by the fatty acids.

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1 , SAPP2 , and SAPP3 . We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

A complex of three plasma proteins, including the high molecular mass kininogen (HK), prekallikrein (PK), and factor XII (FXII), is known to assemble on cell surfaces to release bradykinin-related proinflammatory peptides (kinins). Only recently, the binding of HK to human macrophages was described in the U-937 cell line model. In the present study, the adsorption of the other components of plasma kinin-generating system to these cells was characterized. FXII was found to tightly bind to U-937 cells and was also shown to partially compete with HK for the same binding sites on the macrophage surface. The Mac-1 and gC1qR proteins were found to be receptors for FXII on the cell surface. PK indirectly docked to the macrophages via the cell-bound HK and FXII. Within the complex of these proteins assembled on the macrophage, PK could be activated by FXII/FXIIa or independently of this factor, and the active PK effectively released kinins from HK. The cell surface-bound HK could also be the substrate for tissue kallikrein approaching the cell from the bulk fluid. The kinins released at the surface are suggested to induce secondary responses in the macrophages, leading to further propagation of the inflammatory state.

Staphylococcus aureus , Staphylococcus epidermidis , and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.