Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins

The cupin superfamily is a group of functionally diverse proteins that are found in all three kingdoms of life, Archaea, Eubacteria, and Eukaryota. These proteins have a characteristic signature domain comprising two histidine- containing motifs separated by an intermotif region of variable length. This domain consists of six beta strands within a conserved beta barrel structure. Most cupins, such as microbial phosphomannose isomerases (PMIs), AraC- type transcriptional regulators, and cereal oxalate oxidases (OXOs), contain only a single domain, whereas others, such as seed storage proteins and oxalate decarboxylases (OXDCs), are bi-cupins with two pairs of motifs. Although some cupins have known functions and have been characterized at the biochemical level, the majority are known only from gene cloning or sequencing projects. In this study, phylogenetic analyses were conducted on the conserved domain to investigate the evolution and structure/function relationships of cupins, with an emphasis on single- domain plant germin-like proteins (GLPs). An unrooted phylogeny of cupins from a wide spectrum of evolutionary lineages identified three main clusters, microbial PMIs, OXDCs, and plant GLPs. The sister group to the plant GLPs in the global analysis was then used to root a phylogeny of all available plant GLPs. The resulting phylogeny contained three main clades, classifying the GLPs into distinct subfamilies. It is suggested that these subfamilies correlate with functional categories, one of which contains the bifunctional barley germin that has both OXO and superoxide dismutase (SOD) activity. It is proposed that GLPs function primarily as SODs, enzymes that protect plants from the effects of oxidative stress. Closer inspection of the DNA sequence encoding the intermotif region in plant GLPs showed global conservation of thymine in the second codon position, a character associated with hydrophobic residues. Since many of these proteins are multimeric and enzymatically inactive in their monomeric state, this conservation of hydrophobicity is thought to be associated with the need to maintain the various monomer- monomer interactions. The type of structure-based predictive analysis presented in this paper is an important approach for understanding gene function and evolution in an era when genomes from a wide range of organisms are being sequenced at a rapid rate.

Leafy, terminal flower1 and agamous are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.

Cusp observations during a sequence of fast IMF B-Z reversals

In recent years, a large number of papers have reported the response of the cusp to solar wind variations under conditions of northward or southward Interplanetary Magnetic Field (IMF) Z-component (BZ). These studies have shown the importance of both temporal and spatial factors in determining the extent and morphology of the cusp and the changes in its location, connected to variations in the reconnection geometry. Here we present a comparative study of the cusp, focusing on an interval characterised by a series of rapid reversals in the BZ-dominated IMF, based on observations from space-borne and ground-based instrumentation. During this interval, from 08:00 to 12:00 UT on 12 February 2003, the IMF BZ component underwent four reversals, remaining for around 30 min in each orientation. The Cluster spacecraft were, at the time, on an outbound trajectory through the Northern Hemisphere magnetosphere, whilst the mainland VHF and Svalbard (ESR) radars of the EISCAT facility were operating in support of the Cluster mission. Both Cluster and the EISCAT were, on occasion during the interval, observing the cusp region. The series of IMF reversal resulted in a sequence of poleward and equatorward motions of the cusp; consequently Cluster crossed the high altitude cusp twice before finally exiting the dayside magnetopause, both times under conditions of northward IMF BZ. The first magnetospheric cusp encounter, by all four Cluster spacecraft, showed reverse ion dispersion typical of lobe reconnection; subsequently, Cluster spacecraft 1 and 3 (only) crossed the cusp for a second time. We suggest that, during this second cusp crossing, these two spacecraft were likely to have been on newly closed field lines, which were first reconnected (opened) at low latitudes and later reconnected again (re-closed) poleward of the northern cusp.

Sequence-selective assembly of tweezer-molecules on linear templates enables frameshift-reading of sequence information

Monomer-sequence information in synthetic copolyimides can be recognised by tweezer-type molecules binding to adjacent triplet-sequences on the polymer chains. In the present paper different tweezer-molecules are found to have different sequence-selectivities, as demonstrated in solution by 1H NMR spectroscopy and in the solid state by single crystal X-ray analyses of tweezer-complexes with linear and macrocyclic oligo-imides. This work provides clear-cut confirmation of polyimide chain-folding and adjacent-tweezer-binding. It also reveals a new and entirely unexpected mechanism for sequence-recognition which, by analogy with a related process in biomolecular information processing, may be termed "frameshift-reading". The ability of one particular tweezer-molecule to detect, with exceptionally high sensitivity, long-range sequence-information in chain-folding aromatic copolyimides, is readily explained by this novel process.

Sequence analysis of the cupin gene family in Synechocystis PCC6803

The recently described cupin superfamily of proteins includes the germin and germinlike proteins, of which the cereal oxalate oxidase is the best characterized. This superfamily also includes seed storage proteins, in addition to several microbial enzymes and proteins with unknown function. All these proteins are characterized by the conservation of two central motifs, usually containing two or three histidine residues presumed to be involved with metal binding in the catalytic active site. The present study on the coding regions of Synechocystis PCC6803 identifies a previously unknown group of 12 related cupins, each containing the characteristic two-motif signature. This group comprises 11 single-domain proteins, ranging in length from 104 to 289 residues, and includes two phosphomannose isomerases and two epimerases involved in cell wall synthesis, a member of the pirin group of nuclear proteins, a possible transcriptional regulator, and a close relative-of a cytochrome c551 from Rhodococcus. Additionally, there is a duplicated, two-domain protein that has close similarity to an oxalate decarboxylase from the fungus Collybia velutipes and that is a putative progenitor of the storage proteins of land plants.

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved beta-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the beta-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase.

Germin and germin-like proteins: evolution, structure, and function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.

Molecular characterization of Iranian wheat stripe virus shows its taxonomic position as a distinct species in the genus Tenuivirus

The full lengths of three genome segments of Iranian wheat stripe virus (IWSV) were amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR) using a primer complementary to tenuivirus conserved terminal sequences. The segments were sequenced and found to comprise 3469, 2337, and 1831 nt, respectively. The gene organization of these segments is similar to that of other known tenuiviruses, each displaying an ambisense coding strategy. IWSV segments, however, are different from those of other viruses with respect to the number of nucleotides and deduced amino acid sequence for each ORF. Depending on the segment, the first 16-22 nt at the 5' end and the first 16 nt at the 3' end are highly conserved among IWSV and rice hoja blanca virus (RHBV), rice stripe virus (RSV) and maize stripe virus ( MStV). In addition, the first 15-18 nt at the 5' end are complementary to the first 16-18 nt at the 3' end. Phylogenetic analyses showed close similarity and a common ancestor for IWSV, RHBV, and Echinochloa hoja blanca virus (EHBV). These findings confirm the position of IWSV as a distinct species in the genus Tenuivirus.

Elusive relationships within order fabales: phylogenetic analyses using matK and rbcL sequence data

The order Fabales, including Leguminosae, Polygalaceae, Quillajaceae and Surianaceae, represents a novel hypothesis emerging from angiosperm molecular phylogenies. Despite good support for the order, molecular studies to date have suggested contradictory, poorly supported interfamilial relationships. Our reappraisal of relationships within Fabales addresses past taxon sampling deficiencies, and employs parsimony and Bayesian approaches using sequences from the plastid regions rbcL (166 spp.) and matK (78 spp.). Five alternative hypotheses for interfamilial relationships within Fabales were recovered. The Shimodaira-Hasegawa test found the likelihood of a resolved topology significantly higher than the one calculated for a polytomy, but did not favour any of the alternative hypotheses of relationship within Fabales. In the light of the morphological evidence available and the comparative behavior of rbcL and matK, the topology recovering Polygalaceae as sister to the rest of the order Fabales with Leguminosae more closely related to Quillajaceae + Surianaceae, is considered the most likely hypothesis of interfamilial relationships of the order. Dating of selected crown clades in the Fabales phylogeny using penalized likelihood suggests rapid radiation of the Leguminosae, Polygalaceae, and (Quillajaceae + Surianaceae) crown clades.