Activity of the natural algicide, cyanobacterin, on eukaryotic microorganisms

Abstract The natural product cyanobacterin has been shown to be toxic to most cyanobacteria at a concentration of approx. 5 μM. We demonstrate here that cyanobacterin will also inhibit the growth of most eukaryotic algae at a similar concentration. Some algae, such as Euglena gracilis , are resistant because they are able to maintain themselves by heterotrophic nutrition. Others, such as Chlamydomonas reinhardtii , can apparently induce a detoxification mechanism to maintain photosynthesis in the presence of low concentrations of the inhibitor. Non-photosynthetic microorganisms are not affected by cyanobacterin.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked beta-solenoid model of HET-s prion fibrils

Fungal prions are infectious filamentous polymers of proteins that are soluble in uninfected cells. In its prion form, the HET-s protein of Podospora anserina participates in a fungal self/non-self recognition phenomenon called heterokaryon incompatibility. Like other prion proteins, HET-s has a so-called "prion domain" (its C-terminal region, HET-s-(218-289)) that is responsible for induction and propagation of the prion in vivo and for fibril formation in vitro. Prion fibrils are thought to have amyloid backbones of polymerized prion domains. A relatively detailed model has been proposed for prion domain fibrils of HET-s based on a variety of experimental constraints (Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., Meier, B. H., Saupe, S. J., and Riek, R. (2005) Nature 435, 844-848). To test specific predictions of this model, which envisages axial stacking of beta-solenoids with two coils per subunit, we examined fibrils by electron microscopy. Electron diffraction gave a prominent meridional reflection at (0.47 nm)(-1), indicative of cross-beta structure, as predicted. STEM (scanning transmission electron microscopy) mass-per-unit-length measurements yielded 1.02 +/- 0.16 subunits per 0.94 nm, in agreement with the model prediction (1 subunit per 0.94 nm). This is half the packing density of approximately 1 subunit per 0.47 nm previously obtained for fibrils of the yeast prion proteins, Ure2p and Sup35p, whence it follows that the respective amyloid architectures are basically different.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

Roles of calcium ions in the activation and activity of the transglutaminase 3 enzyme

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 microm (DeltaH = -6.70 +/- 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.     

Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.     

Filaments of the Ure2p prion protein have a cross-beta core structure

Formation of filaments by the Ure2 protein constitutes the molecular mechanism of the [URE3] prion in yeast. According to the "amyloid backbone" model, the N-terminal asparagine-rich domains of Ure2p polymerize to form an amyloid core fibril that is surrounded by C-terminal domains in their native conformation. Protease resistance and Congo Red binding as well as beta-sheet content detected by spectroscopy-all markers for amyloid-have supported this model, as has the close resemblance between 40 A N-domain fibrils and the fibrillar core of intact Ure2p filaments visualized by cryo-electron microscopy and scanning transmission electron microscopy. Here, we present electron diffraction and X-ray diffraction data from filaments of Ure2p, of N-domains alone, of fragments thereof, and of an N-domain-containing fusion protein that demonstrate in each case the 4.7A reflection that is typical for cross-beta structure and highly indicative of amyloid. This reflection was observed for specimens prepared by air-drying with and without sucrose embedding. To confirm that the corresponding structure is not an artifact of air-drying, the reflection was also demonstrated for specimens preserved in vitreous ice. Local area electron diffraction and X-ray diffraction from partially aligned specimens showed that the 4.7A reflection is meridional and therefore the underlying structure is cross-beta.     

Is the prion domain of soluble Ure2p unstructured?

The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.     

Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 Å.Glycosyltransferases constitute a large family of enzymes that catalyze the transfer of sugar moieties from donor molecules to specific acceptor molecules. Unlike other enzyme families that usually share conserved features in their primary sequences, glycosyltransferases can have highly diversified sequences that have been grouped into more than 90 families (designated GTn, where n = 1, 2, …) (http://www.CAZy.org) (1, 15). However, two families, GT2 and GT4, account for about half of the total number of glycosyltransferases. Despite the large variation in the primary sequences of glycosyltransferases, their three-dimensional structures are usually conserved. There are two major glycosyltransferase structural types, named GT-A and GT-B. The GT-A members contain a single nucleotide-binding domain consisting of six parallel β-strands flanked by connecting α-helices (referred to as a “Rossmann fold” in most of the literature on these enzymes and herein). GT-A enzyme activities are usually metal ion dependent. The GT-B type glycosyltransferases have two Rossmann folds separated by a cleft that forms the substrate-binding site. Metal ions are normally not required for GT-B function. Based on their catalytic mechanism, glycosyltransferases are also classified as either retaining or inverting enzymes depending on the geometry between the sugar donor and the receptor in the product molecule (e.g., depending on whether the anomeric carbon atom is linked to the acceptor via its α or β position). If the anomeric carbon atom has the same configuration in the donor and in the product, the enzyme is classified as a retaining enzyme; if the configurations are different, the enzyme is considered to be an inverting enzyme (2).Many viruses, especially those that infect eukaryotic cells, have extensively glycosylated structural proteins. Glycans coating viral structural proteins serve multiple biological roles, e.g., they mimic host glycans to evade host cell immune reactions, aid in folding or assembly of viral structural proteins, function as a receptor recognized by cell surface proteins, or aid in stabilizing viral particles (see, e.g., reference 36).Typically, viruses use host-encoded glycosyltransferases and glycosidases located in the endoplasmic reticulum (ER) and Golgi apparatus to add and remove N-linked sugar residues from virus glycoproteins either during or shortly after translation of the protein. This posttranslational processing aids in protein folding and requires other host-encoded enzymes. After folding and assembly, virus glycoproteins are transported by host-sorting and membrane transport functions to virus-specified regions in host membranes, where they displace host glycoproteins. Progeny viruses then bud through these virus-specific target membranes, in what is usually the final step in the assembly of infectious virions (3, 14, 21, 36). Thus, nascent viruses become infectious only by budding through the target membrane, usually the plasma membrane, as they are released from the cell. Consequently, the glycan portion of virus glycoproteins is host specific. The theme that emerges is that virus glycoproteins are synthesized and glycosylated by the same mechanisms as host glycoproteins. Therefore, the only way to alter glycosylation of virus proteins is to either grow the virus in a different host or have a mutation in the virus protein that alters the protein glycosylation site.One explanation for this scenario is that, in general, viruses lack genes encoding glycosyltransferases. However, a few virus-encoded glycosyltransferases have been reported in recent years (see reference 17 for a review). Often these virus-encoded glycosyltransferases add sugars to compounds other than proteins. For instance, some phage-encoded glycosyltransferases modify virus DNA to protect it from host restriction endonucleases (see, e.g., reference 10), and a glycosyltransferase encoded by baculoviruses modifies a host insect ecdysteroid hormone, leading to its inactivation (22). Bovine herpesvirus 4 encodes a β-1,6-N-acetyl-glucosaminyltransferase that is localized in the Golgi apparatus and is probably involved in posttranslational modification of the virus structural proteins (32).One group of viruses differs from the scenario that viruses use the host machinery located in the ER and the Golgi apparatus to glycosylate their glycoproteins. These viruses are the large, plaque-forming, double-stranded DNA (dsDNA)-containing chloroviruses (family Phycodnaviridae) that infect eukaryotic algae (4, 34, 39, 40). The chloroviruses have up to 400 protein-encoding genes (or coding sequences [CDSs]). Annotation of six chlorovirus genomes showed that each virus encodes 3 to 6 putative glycosyltransferases (7-9, 16, 33). Three of these viruses, NY-2A, AR158, and the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1), infect Chlorella strain NC64A. Two of the viruses, MT325 and FR483, infect Chlorella Pbi, and one of them, Acanthocystis turfacea chlorella virus (ATCV-1), infects Chlorella SAG 3.83.Glycosylation of the PBCV-1 major capsid protein, Vp54, is at least partially performed by the viral glycosyltransferases (11, 20, 33, 38, 41). PBCV-1 encodes 5 putative glycosyltransferases. A previous structural study established that the N-terminal 211 amino acids of the A64R protein from PBCV-1 form a GT-A group glycosyltransferase that is a retaining enzyme belonging to the GT34 family and that UDP-glucose possibly serves as the donor sugar (41).Among the four additional PBCV-1 glycosyltransferase-encoding genes, gene a546l encodes a 396-amino-acid protein that resembles members in the GT4 family of glycosyltransferases, based on amino acid sequence comparison of members in the CAZy classification (1, 15). Homologs of this protein, A546L, are encoded by 3 other chloroviruses, NY-2A, AR158, and ATCV-1. Here, we report the crystal structure of one of these homologs, B736L, at 2.3-Å resolution.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.