Volume 384, Issue 7

Lipid modification of eukaryotic proteins by protein prenyltransferases is required for critical signaling pathways, cell cycle progression, cytoskeleton remodeling, induction of apoptosis and vesicular trafficking. This review analyzes the influence of distinct states of sequential posttranslational processing that can be obtained after single or double prenylation, reversible palmitoylation, proteolytic cleavage of the C-terminus and possible reversible carboxymethylation. This series of modifications, as well as the exact length of the prenyl anchor, are determinants in protein-membrane and specific protein-protein interactions of protein prenyltransferase substrates. Furthermore, the occurrence and distribution of pseudogenes of protein prenyltransferase subunits are discussed. Besides being developed as anti-cancer agents, prenyltransferase inhibitors are effective against an increasing number of parasitic diseases. Extensive screens for protein prenyltransferases in genomic data of fungal and protozoan pathogens unveil a series of new pharmacologic targets for prenyltransferase inhibition, including the parasites Brugia malayi, Onchocerca volvulus, Aspergillus nidulans, Pneumocystis carinii, Entamoeba histolytica, Strongyloides stercoralis, Trichinella spiralis and Cryptosporidium parvum.

Oxidative stress evokes various cellular events, including activation of transcription factors, apoptosis, and cell cycle arrest. Accumulating evidence shows that reduction/oxidation (redox) plays an important role in the regulation of apoptosis and cell cycle arrest elicited by oxidative stress. Cellular redox is controlled by the thioredoxin (TRX) and glutathione (GSH) systems. TRX and GSH systems regulate cell growth and cell death by the activation of transcription factors, the sensitivity of cells to cytokines and growth factors, and the components of the apoptosis pathways. This brief review describes the current knowledge on the redox regulation of cell growth and apoptosis.

Binding of flavan-3-ols to nuclei is characteristic of Tsuga canadensis (coniferous tree). This is achieved with the DMACA reagent (ρ-dimethylaminocinnamaldehyde), which stains almost exclusively monomeric and oligomeric flavan-3-ols with an intense blue colour. Deep flavanol staining also occurred when calf cells of small intestine were enriched with added catechins. In order to detect the components of nuclei that may associate with catechins, the principal components of chromatin (DNA, histones) were subjected to UV-VIS spectroscopic titration. DNA or histone sulphate containing the histones H1, H2A, H2B, H3 and H4 were dissolved in cationic and anionic buffers (Tris, phosphate) at different pH values (pH 8.0, 7.4 and 7.0) and titrated with EGCG (epigallocatechin gallate) or catechin. The results show that DNA of calf thymus and the catechins investigated form no spectroscopically detectable association equilibria. However, strong association complexes are found between histone sulphate and EGCG or catechin by means of the Mauser diagrams (A and AD diagrams). The association equilibria can be accompanied by aggregation (precipitation) of histone proteins, especially initiated by EGCG. The titration equilibria are spectroscopically more pronounced in Tris buffers at higher pH values than at lower values, whereas in phosphate buffers the opposite trend is found. Surprisingly, catechin shows nearly no interactions with histone sulphate in phosphate buffers in the pH range 7.0-8.0, which is in contrast to EGCG. Fundamentally, the targets of chromosomes for catechins seem to be the histone proteins of chromatin.

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'- UTR, or sandwiched between the 5' and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.

Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitztype serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the presence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demonstrate that DNA demethylation by 5'-aza-2'-deoxycytidine restores TFPI-2 transcription in JAR choriocarcinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI-2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor trophoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silencing and that the TFPI-2 gene may be a candidate tumor suppressor gene.

The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate 4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme Oacetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble Oacetyltransferase maximally operated at 30C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 M for AcCoA and 1.2 M for GD3. CoA inhibits the enzyme with a Ki value of 14.8 M. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.

Extracellular liquid of the edible fungus Lepista irina was found to effectively degrade β,β-carotene. β-Ionone, β-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed as volatile breakdown products of β,β-carotene with mycelium-free culture supernatants, whereas β-apo 10'-carotenal was identified as non-volatile degradation product. The key enzyme catalyzing the oxidative cleavage of β,β-carotene was purified with an overall yield of 63% and a purification factor of 43. Biochemical characterization showed a molecular mass of 50.5 kDa and an isoelectric point of 3.75. Fastest β,β-carotene degradation occurred at 34C and pH values between 3.5 and 4. Degenerate oligonucleotides were derived from N-terminal and internal amino acid sequences. By means of PCRbased cDNA-library screening a 1284 bp cDNA was identified which showed great overall similarity to Pleurotus eryngii polyvalent peroxidases. The obtained sequence contains an open reading frame of 1083 nucleotides, encoding a polypeptide of 361 amino acids. A 30 amino acid signal peptide was identified upstream of the Nterminal sequence of the mature enzyme. The L. irina versatile peroxidase represents the first microbial enzyme capable of carotenoid degradation that has been characterized on a molecular level, proving the participation of extracellular enzymes of white rot fungi in biotic carotenoid degradation processes.

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.

Several activators of bovine leukemia virus (BLV) expression, including lipopolysaccharides, phorbol esters and calcium ionophores, are known to generate reactive oxygen species (ROS). Therefore the influence of H2O2 on BLV expression in two BLV producing cell lines was investigated. The effect of H2O2 on BLV expression is apparently dose-dependent. Incubation of FLK/BLV cells with low concentrations of H2O2 (2.5 to 10 uM) induced a marked enhancement of BLV p24 synthesis and an activation of the long terminal repeat (LTR). Higher concentrations resulted in a decrease of proliferation, induction of apoptosis and in a decrease of BLV synthesis. Furthermore, in both cell lines H2O2 treatment led to the activation of NF-κB. Pretreatment of cells with antioxidants abrogated the H2O2-induced BLV expression. Taken together, our findings suggest that oxidative stress stimulates BLV expression via activation of NF-κB, raising the possibility that biological sources of H2O2, such as stimulated phagocytes, may influence BLV expression.

During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin αvβ3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized αvβ3-directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of αvβ3, and was accompanied by rapid and transient tyrosinephosphorylation of p44erk 1/p42erk 2 mitogen-activated protein kinase. Moreover, overexpression of αvβ3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon αvβ3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of cmyc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-α (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of human ovarian cancer cells.

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ 6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (NhTIMP-1-chCysWT, N-hTIMP-1- chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.

Mouse polyclonal antibodies against placental cysteine proteinase inhibitor (CPI) react with the placental 67 kDa CPI on Western blots, and CPI present in ovarian cancer homogenate and serum was shown by double immunodiffusion to react with the same antiserum. By immunohistochemical staining, positive expression of high molecular weight CPI was observed on the tumour cell surface in serous and endometrioid ovarian carcinomas with metastasis. Normal endometrioid tissue was not stained with anti-placental CPI antibodies. Cathepsin B and procathepsin B median levels in ovarian cancer tissue homogenates increased progressively with FIGO stage of the disease. The enzyme level decreased 22-fold after treatment of tissue homogenates with 5 nM purified CPI. These results provide evidence that addition of CPI reduces the levels of cysteinetype cathepsins to those of normal non-cancerous values.

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wildtype protease agree well with published data for cloned HIV-protease. The enzymatic test was evaluated with recombinant HIV-protease derived from eight HIV-positive patients scored from sensitive to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.