ADAM and ADAMTS family proteins and their role in the colorectal cancer etiopathogenesis

The ADAM and ADAMTS families, also called adamalysins belong to an important group of extracellular matrix proteins. The ADAMs family belong to both the transmembrane and secreted proteins, while ADAMTS family only contains secreted forms. Adamalysins play an important role in the cell phenotype regulation via their activities in signaling pathways, cell adhesion and migration. The human proteome contains 21 ADAM, and 19 ADAMTS proteins, which are involved in extracellular matrix remodeling, shedding of various substrates such as: adhesion ligands, growth factors, their receptors and diverse cytokines. Recent studies provide evidence that adamalysins play a crucial role in colorectal cancer (CRC) etiopathogenesis. It seems possible that adamalysins might be used as CRC prediction markers or potential pharmaceutical targets. [BMB Reports 2013; 46(3): 139-150]     

Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) Proteins Function as the Human IL-8-Binding Effectors and Contribute to Pathogen Entry into Human Neutrophils

Mycobacterium tuberculosis is an extremely successful intracellular pathogen that has evolved a broad spectrum of pathogenic mechanisms that enable its manipulation of host defense elements and its survival in the hostile environment inside phagocytes. Cellular influx into the site of mycobacterial entry is mediated by a variety of chemokines, including interleukin-8 (IL-8), and the innate cytokine network is critical for the development of an adaptive immune response and infection control. Using affinity chromatography, liquid chromatography electrospray ionization tandem mass spectrometry and surface plasmon resonance techniques, we identified M. tuberculosis AtsG arylsulphatase, bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyl transferase (GlmU) and S-adenosyl-L-homocysteine hydrolase (SahH) as the pathogen proteins that bind to human IL-8. The interactions of all of the identified proteins (AtsG, GlmU and SahH) with IL-8 were characterized by high binding affinity with K_D values of 6.83x10^-6 M, 5.24x10^-6 M and 7.14x10^-10 M, respectively. Furthermore, the construction of Mtb mutant strains overproducing AtsG, GlmU or SahH allowed determination of the contribution of these proteins to mycobacterial entry into human neutrophils. The significantly increased number of intracellularly located bacilli of the overproducing M. tuberculosis mutant strains compared with those of “wild-type” M. tuberculosis and the binding interaction of AtsG, GlmU and SahH proteins with human IL-8 may indicate that these proteins participate in the modulation of the early events of infection with tubercle bacilli and could affect pathogen attachment to target cells.     

Latency,thermal stability,and identification of an inhibitory compound of mirolysin,a secretory protease of the human periodontopathogen Tannerella forsythia

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a “cysteine-switch” mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (K_i = 3.2 µM), binding to the S1′ subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.     

Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a “key and lock” interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.Malignant transformation is universally accompanied by changes in cell surface glycosylation. A glycolipid, GD2 ganglioside (GD2)¹, is one of the most prominent tumor-associated antigens, ranking in the 12th position of the NCI prioritized list of cancer vaccine targets (1). GD2 is embedded in the outer plasma membrane with its ceramide tail (fatty acid coupled sphingosine). The sugar moiety is exposed to the extracellular milieu and is composed of glucose (Glc; linked to ceramide), galactose (Gal) and N-acetylgalactosamine (GalNAc). Two additional sialic acid residues (N-acetylneuraminic acid, NeuAc) branch form Gal and provide GD2 with a negative charge (Fig. 1). Overexpression of GD2 is well documented in neuroblastoma, melanoma, certain osteosarcomas, small cell lung cancers, and soft tissue sarcomas (2–4).Open in a separate windowFig. 1.Recognition of GD2 ganglioside by monoclonal antibody 14G2a at the cell surface. (top panel) Antigen combining region of 14G2a antibody recognizes the sugar moiety of GD2 ganglioside (yellow), which is exposed to the extracellular milieu. The lipid part of the ganglioside is buried inside the cell membrane. GD2 bound Fab structure determined in this study is shown in color. Fc fragment (PDB ID: 1igt) and membrane model derived from published data are shown in corresponding scale and colored gray. (bottom panel) Chemical structure of GD2 ganglioside and sugar ring nomenclature used throughout the study.The concept of therapeutic targeting of GD2 is currently most advanced in neuroblastoma, the most common extracranial tumor of childhood. Neuroblastoma is a heterogenous and complex disease. Spontaneous remissions are sometimes observed, but more than a half of the patients are diagnosed with a high-risk neuroblastoma of poor prognosis. This highlights the demand for treatment modalities that would offer major clinical benefits for this group of patients (5). High and stable presence of GD2 on cancer cells in neuroblastoma and limited expression on relevant normal tissues (i.e. neurons, peripheral nerve fibers and skin melanocytes) allows diagnosis, detection of metastases, treatment monitoring and, most importantly, targeting of the tumor itself.GD2-specific monoclonal antibodies have been extensively tested in clinics. This includes a mouse 14G2a antibody (IgG2a; derived from a mouse 14.18 antibody of IgG3 subclass), and improved modifications thereof including a chimeric antibody ch14.18, and recently a humanized antibody hu14.18K322A. Moreover, mouse 3F8 antibody (IgG3) and recently its humanized derivative hu3F8 were also evaluated. The antibodies were demonstrated to engage antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against neuroblastoma (5). Additionally, direct cytotoxic effects were observed in neuroblastoma models (6). The results of a randomized clinical trial published in 2010, evaluating ch14.18, interleukin-2 and granulocyte and macrophage-colony stimulating factor combined with a standard maintenance agent 13-cis retinoic acid demonstrated significant improvement of outcome in high-risk neuroblastoma patients (7). Based on these and further findings, the Food and Drug Administration (FDA) has just recently approved Unituxin (dinutuximab; ch14.18) combination therapy for high risk neuroblastoma (8). Therefore, the standard care treatment protocols may now be extended with monoclonal antibodies targeting GD2 for a better expected outcome.Antibodies against gangliosides other than GD2 are considered as potential therapeutic agents in different types of cancer. Ganglioside-specific antibodies are moreover involved in various types of autoimmune diseases (9). Nevertheless, the molecular mechanism of ganglioside recognition remains unknown because not a single crystal structure of antibody–ganglioside complex has been determined to date. In particular, it is not known how the specificity against GD2 is achieved in antibodies evaluated in clinics. Although crystal structures of empty ME36.1 antibody specific for GD2 and GD3 (10) and empty 3F8 antibody specific for GD2 (11) were determined, the conclusions concerning GD2 binding have to be treated with caution because of general limitations in reliable prediction of binding modes of complex, flexible ligands in dynamic pockets.The success of GD2-specific antibodies in treatment of neuroblastoma fuels investigation on active immunization strategies. To overcome poor antigenicity of GD2, glycolipid surrogates including peptide mimetics are being developed. The idea of a peptide vaccine eliciting anticarbohydrate response has been precedented in the case of Group B Streptococcus polysaccharide (12). Multiple peptides mimicking GD2 in its binding to specific antibodies were selected using phage display (13, 14) and some have been demonstrated to elicit protective, GD2 directed response in preclinical studies. However, the structural basis of peptide-ganglioside mimicry and its relation to the potential of particular peptides to induce GD2 directed immune response remain unknown.Here, we analyze the interactions guiding ganglioside recognition by an antibody and the structural basis of peptide-ganglioside mimicry. The crystal structure of Fab fragment of 14G2a antibody in a complex with the sugar moiety of GD2 ganglioside is provided and the binding mode is discussed in detail. Structure of an empty 14G2a antibody is reported for reference. The major conclusions are verified by directed mutagenesis and antibody variant with increased affinity toward GD2 is developed using structure guided approach. The binding modes of two largely divergent peptide mimics of GD2 (15) at the antigen-binding site of 14G2a antibody are reported and compared with that of the carbohydrate. Mouse 14G2a antibody was chosen for this study because it contains the same antigen binding region as the ch14.18 chimeric antibody recently approved by FDA (8).     

The arbuscular mycorrhizal Paraglomus majewskii sp. nov. represents a distinct basal lineage in Glomeromycota

Paraglomus majewskii sp. nov. (Glomeromycota) is described and illustrated. It forms single spores, which are hyaline through their life cycle, globose to subglobose, (35-)63(-78) μm diam, sometimes egg-shaped, 50-70 × 65-90 μm, and have an unusually narrow, (3.2-)4.6(-5.9) μm, cylindrical to slightly flared subtending hypha. The spore wall of P. majewskii consists of an evanescent, short-lived outermost layer, a laminate middle layer, and a flexible innermost layer, which adheres tightly to the middle layer. None of the spore wall layers stain in Melzer's reagent. In single-species cultures with Plantago lanceolata as the host plant P. majewskii formed arbuscular mycorrhizae staining violet in trypan blue. P. majewskii has been isolated from several, distant geographic regions and from different habitats. In phylogenetic analyses of partial nrDNA SSU and LSU sequences the fungus formed mono-phyletic group with Paraglomus species; however it represents a well separated distinct lineage. Its nrDNA sequences are highly similar to in planta arbuscular mycorrhizal fungal sequences from different habitats in Spain and Ecuador.     

Arabidopsis BPG2: a phytochrome-regulated gene whose protein product binds to plastid ribosomal RNAs

BPG2 (Brz-insensitive pale green 2) is a dark-repressible and light-inducible gene that is required for the greening process in Arabidopsis. Light pulse experiments suggested that light-regulated gene expression of BPG2 is mediated by phytochrome. The T-DNA insertion mutant bpg2-2 exhibited a reduced level of chlorophyll and carotenoid pigmentation in the plastids. Measurements of time resolved chlorophyll fluorescence and of fluorescence emission at 77 K indicated defective photosystem II and altered photosystem I functions in bpg2 mutants. Kinetic analysis of chlorophyll fluorescence induction suggested that the reduction of the primary acceptor (QA) is impaired in bpg2. The observed alterations resulted in reduced photosynthetic efficiency as measured by the electron transfer rate. BPG2 protein is localized in the plastid stroma fraction. Co-immunoprecipitation of a formaldehyde cross-linked RNA-protein complex indicated that BPG2 protein binds with specificity to chloroplast 16S and 23S ribosomal RNAs. The direct physical interaction with the plastid rRNAs supports an emerging model whereby BPG2 provides light-regulated ribosomal RNA processing functions, which are rate limiting for development of the plastid and its photosynthetic apparatus.     

On the simple random-walk models of ion-channel gate dynamics reflecting long-term memory

Several approaches to ion-channel gating modelling have been proposed. Although many models describe the dwell-time distributions correctly, they are incapable of predicting and explaining the long-term correlations between the lengths of adjacent openings and closings of a channel. In this paper we propose two simple random-walk models of the gating dynamics of voltage and Ca(2+)-activated potassium channels which qualitatively reproduce the dwell-time distributions, and describe the experimentally observed long-term memory quite well. Biological interpretation of both models is presented. In particular, the origin of the correlations is associated with fluctuations of channel mass density. The long-term memory effect, as measured by Hurst R/S analysis of experimental single-channel patch-clamp recordings, is close to the behaviour predicted by our models. The flexibility of the models enables their use as templates for other types of ion channel.     

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolvingactivity of Synechocystis PCC6803 pgsA mutant cells. Shortageof PG led to decreased photosynthetic activity, which, similarto the effect of high light exposure, is likely to generatethe production of reactive oxygen species (ROS) or free radicals.Protection of the PG-depleted cells against light-induced damageincreased the echinenone and myxoxanthophyll content of thecells. The increased carotenoid content was localized in a solublefraction of the cells as well as in isolated thylakoid and cytoplasmicmembranes. The soluble carotenoid fraction contained carotenederivatives, which may bind to proteins. These carotene–proteincomplexes are similar to orange carotenoid protein that is involvedin yielding protection against free radicals and ROS. An increasein the content of myxoxanthophyll and echinenone upon PG depletionsuggests that PG depletion regulates the biosynthetic pathwayof specific carotenoids.