PP2A: the expected tumor suppressor

PP2A is one of the few serine/threonine-specific phosphatases in the cell, and its complex structure and regulation guarantees its many different functions. Some viruses have chosen to target this enzyme system in order to manage the host cell machinery for their own profit and to program cells into a malignant state. Suppression of PR61/B'gamma, a specific third regulatory subunit of PP2A, can substitute for the viral SV40 protein small t antigen in causing tumorigenic transformation of several human cell lines -- provided that telomerase, SV40 large T antigen and oncogenic Ras are also present. Accumulation of c-Myc seems to be the common denominator.     

Stimulation of TNF-alpha release by fungal cell wall polysaccharides

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.     

The imbalance between metalloproteinases and their tissue inhibitors is involved in the pathogenesis of dermatitis herpetiformis

Dermatitis herpetiformis (DH) is a subepidermal autoimmune disease characterized by skin and intestinal lesions consistent with coeliac disease. There are also some data that metalloproteinases (MMPs) are involved in the development of skin lesions in DH, however their exact role in this process is not fully understood. The aim of the study was to investigate whether MMPs and their inhibitors are involved in pathogenesis of DH. Skin biopsies were taken from 13 patients with active DH and from 10 healthy subjects. The localization and expression of MMPs and TIMPs were examined by immunohistochemistry. MMPs expression was detected in basal keratinocytes and in the whole epidermis in all of the DH subjects. Neutrophils in microabscesses and in blister fluid were also positive for MMPs. Expression of TIMPs was moderate or weak in all examined biopsies. Our results allow us to conclude that imbalance between these enzymes takes an important role in the pathogenesis of DH.     

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.     

Probing the mechanism of the Archaeoglobus fulgidus inositol-1-phosphate synthase

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate. In the sulfate-reducing archaeon Archaeoglobus fulgidus it is a metal-dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-inositol-1,1'-phosphate. Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS. Six charged residues in the active site (Asp225, Lys274, Lys278, Lys306, Asp332, and Lys367) and two noncharged residues (Asn255 and Leu257) have been changed to alanine. The charged residues are located at the active site and were proposed to play binding and/or direct catalytic roles, whereas noncharged residues are likely to be involved in proper binding of the substrate. Kinetic studies showed that only N255A retains any measurable activity, whereas two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD+ to NADH. The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31P line width of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD+ (detected via changes in the protein intrinsic fluorescence). Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.     

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

Amplification of telomeric arrays via rolling-circle mechanism

Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.     

Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.