The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.     

<Emphasis Type="Italic">Shewanella oneidensis</Emphasis>: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background  

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H₂ under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.     

Zippers Make Signals: NCAM-mediated Molecular Interactions and Signal Transduction

The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell–cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitroand in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.Special issue dedicated to Lawrence F. Eng.     

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.     

Integration and Fixation Preferences of Human and Mouse Endogenous Retroviruses Uncovered with Functional Data Analysis

Endogenous retroviruses (ERVs), the remnants of retroviral infections in the germ line, occupy ~8% and ~10% of the human and mouse genomes, respectively, and affect their structure, evolution, and function. Yet we still have a limited understanding of how the genomic landscape influences integration and fixation of ERVs. Here we conducted a genome-wide study of the most recently active ERVs in the human and mouse genome. We investigated 826 fixed and 1,065 in vitro HERV-Ks in human, and 1,624 fixed and 242 polymorphic ETns, as well as 3,964 fixed and 1,986 polymorphic IAPs, in mouse. We quantitated >40 human and mouse genomic features (e.g., non-B DNA structure, recombination rates, and histone modifications) in ±32 kb of these ERVs’ integration sites and in control regions, and analyzed them using Functional Data Analysis (FDA) methodology. In one of the first applications of FDA in genomics, we identified genomic scales and locations at which these features display their influence, and how they work in concert, to provide signals essential for integration and fixation of ERVs. The investigation of ERVs of different evolutionary ages (young in vitro and polymorphic ERVs, older fixed ERVs) allowed us to disentangle integration vs. fixation preferences. As a result of these analyses, we built a comprehensive model explaining the uneven distribution of ERVs along the genome. We found that ERVs integrate in late-replicating AT-rich regions with abundant microsatellites, mirror repeats, and repressive histone marks. Regions favoring fixation are depleted of genes and evolutionarily conserved elements, and have low recombination rates, reflecting the effects of purifying selection and ectopic recombination removing ERVs from the genome. In addition to providing these biological insights, our study demonstrates the power of exploiting multiple scales and localization with FDA. These powerful techniques are expected to be applicable to many other genomic investigations.     

Microsatellite Interruptions Stabilize Primate Genomes and Exist as Population-Specific Single Nucleotide Polymorphisms within Individual Human Genomes

Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation. However, human polymorphism levels at interrupted microsatellites (iMSs) are not known at a genome-wide scale, and the pathways for gaining interruptions are poorly understood. Using the 1000 Genomes Phase-1 variant call set, we interrogated mono-, di-, tri-, and tetranucleotide repeats up to 10 units in length. We detected ∼26,000–40,000 iMSs within each of four human population groups (African, European, East Asian, and American). We identified population-specific iMSs within exonic regions, and discovered that known disease-associated iMSs contain alleles present at differing frequencies among the populations. By analyzing longer microsatellites in primate genomes, we demonstrate that single interruptions result in a genome-wide average two- to six-fold reduction in microsatellite mutability, as compared with perfect microsatellites. Centrally located interruptions lowered mutability dramatically, by two to three orders of magnitude. Using a biochemical approach, we tested directly whether the mutability of a specific iMS is lower because of decreased DNA polymerase strand slippage errors. Modeling the adenomatous polyposis coli tumor suppressor gene sequence, we observed that a single base substitution interruption reduced strand slippage error rates five- to 50-fold, relative to a perfect repeat, during synthesis by DNA polymerases α, β, or η. Computationally, we demonstrate that iMSs arise primarily by base substitution mutations within individual human genomes. Our biochemical survey of human DNA polymerase α, β, δ, κ, and η error rates within certain microsatellites suggests that interruptions are created most frequently by low fidelity polymerases. Our combined computational and biochemical results demonstrate that iMSs are abundant in human genomes and are sources of population-specific genetic variation that may affect genome stability. The genome-wide identification of iMSs in human populations presented here has important implications for current models describing the impact of microsatellite polymorphisms on gene expression.     

Fe‐N‐C Oxygen Reduction Fuel Cell Catalyst Derived from Carbendazim: Synthesis,Structure, and Reactivity

New non‐PGM catalysts from the family of Fe‐N‐C pyrolyzed materials are reported. They are synthesized using a templating silica powder with iron nitrate and carbendazim (CBDZ) precursors (sacrificial support method). The synthesis involves high temperature pyrolysis, followed by etching of the sacrificial support (silica) and obtaining a “self‐supported” open frame morphology catalyst. Both the temperature of heat treatment and Fe to CBDZ ratio play a crucial role in the final catalytic activity in oxygen reduction reaction (ORR). Prepared materials have extremely high durability in RDE tests, ending up with more than 94% of initial activity (by E_1/2 value) after 10 000 cycles in an oxygen atmosphere, which is the result we report for the first time. Evaluation of these new M‐N‐C catalysts in a single membrane electrode assembly (MEA) has shown an exceptionally high open circuit voltage (OCV) of 1 V and the world's second best performance with no IR correction. MEA tests have shown high current density of 700 mA cm^‐2 at 0.6 V and 120 mA cm^‐2 at 0.8 V. In‐depth structure‐to‐property correlation presents an evidence that Fe‐N_x centers are the active sites playing a key role in oxygen reduction reaction.     

Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth But Not of Photosynthesis in Arabidopsis

Mitogen-activated protein kinase （MAPK） pathways regulate signal transduction from different cellular com- partments and from the extracellular environment to the nucleus in all eukaryotes. One of the best-characterized MAPKs in Arabidopsis thaliana is MPK4, which was shown to be a negative regulator of systemic-acquired resistance. The mpk4 mutant accumulates salicylic acid （SA）, possesses constitutive expression of pathogenesis-related （PR） genes, and has an extremely dwarf phenotype. We show that suppression of SA and phylloquinone synthesis in chloroplasts by knocking down the IC51 gene （by crossing it with the icsl mutant） in the mpk4 mutant background did not revert mpk4-impaired growth. However, it did cause changes in the photosynthetic apparatus and severely impaired the quantum yield of pho- tosystem Ih Transmission microscopy analysis revealed that the chloroplasts＇ structure was strongly altered in the mpk4 and mpk4/icsl double mutant. Analysis of reactive oxygen species （ROS）-scavenging enzymes expression showed that suppression of SA and phylloquinone synthesis in the chloroplasts of the mpk4 mutant caused imbalances in ROS homeo- stasis which were more pronounced in mpk4/icsl than in mpk4. Taken together, the presented results strongly suggest that MPK4 is an ROS/hormonal rheostat hub that negatively, in an SA-dependent manner, regulates immune defenses, but at the same time positively regulates photosynthesis, ROS metabolism, and growth. Therefore, we concluded that MPK4 is a complex regulator of chloroplastic retrograde signaling for photosynthesis, growth, and immune defenses in Arabidopsis.     

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the β-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2′,3′-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn²⁺ and Ni²⁺ for catalysis, whereas Zn²⁺ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn²⁺ ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn²⁺ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.In many environments inorganic phosphate, an essential nutrient, can fall to extremely low concentrations, forcing microorganisms to utilize other forms of phosphorus to survive. In such cases, organophosphonates can comprise a major fraction of the total phosphorus available to biological systems (e.g. 2-aminoethylphosphonate is a widespread natural product). However, cleavage of the highly stable carbon-phosphorus (CP)⁵ bond to release inorganic phosphate requires specialized enzymes. One such enzyme activity found widely in bacteria is CP-lyase (1). Cleavage of the CP bond of organophosphonates by CP-lyase yields inorganic phosphate and, remarkably, a hydrocarbon. CP-lyase is actually a multienzyme system, encoded by the phn operon (phnCDEFGHIJKLMNOP), which is induced by low concentrations of phosphate as part of the pho regulon. Gene deletion studies in Escherichia coli have shown that phnGHIJKLM are essential for catalysis of CP bond cleavage, whereas the remaining genes probably encode transport, regulatory, or accessory functions (2). Only a handful of the proteins encoded by the phn operon have been characterized to date. PhnD was shown to be a periplasmic binding protein with high affinity for organophosphonates (3); the three-dimensional structure of PhnH, one of the proteins essential for CP-lyase catalysis, was recently solved, but a function has yet to be determined (4); PhnN was shown to be an ATP-dependent kinase that provides a redundant pathway to 5-phospho-d-ribofuranosyl-α-1-diphosphate (5); and PhnO was demonstrated to be an acetyl-CoA-dependent N-acyltransferase with activity toward a wide range of aminoalkylphosphonates (6).Although the phnP gene is not essential for CP bond cleavage by cells in liquid culture (2), cell growth on solid media supplemented with methylphosphonate or phosphite as the sole phosphorus source is prevented by phnP mutations (7), suggesting a critical regulatory or accessory role for PhnP. Accordingly, phnP appears frequently in the phn operon in various species of bacteria, typically following the phnN gene (8). PhnP is predicted based on its sequence to be a member of the β-lactamase family of metal-dependent hydrolases with greatest homology to enzymes from the tRNase Z (ProDom family PD352433) and ElaC families (9), the latter erroneously annotated as composed of arylsulfatases but later determined to also belong to the tRNase Z family (10, 11). The tRNase Z enzymes are endonucleases used by prokaryotes and eukaryotes to cleave a specific phosphodiester bond near the 3′-end of pre-tRNA, yielding a 3′-end that can be coupled to an amino acid. These enzymes typically use two active site bound Zn²⁺ ions to simultaneously lower the pK_a of a nucleophilic water molecule and stabilize negative charge development on the phosphodiester linkage undergoing nucleophilic attack (12). Since it is not clear how a tRNase activity would support cell growth with an organophosphonate as a sole phosphorus source, we set out to characterize the substrate specificity and three-dimensional structure of PhnP to learn more about this critical CP-lyase enzyme.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.