Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

Background

In eukaryotes, PPP (p rotein p hosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases.

Results

We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhi zobiales / Rh odobacterales / Rh odospirillaceae-l ike ph osphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed She wanella-l ike ph osphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed A paH-l ike ph osphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs.

Conclusions

Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.

     

Expression of sorghum gene <Emphasis Type="Italic">SbSGL</Emphasis> enhances grain length and weight in rice

Grain weight is a major determining factor of rice (Oryza sativa L.) yield and the comprehensive embodiment of grain length, width, and thickness. Here, we describe the molecular and functional characterization of SbSGL (Sorghum bicolor L. stress tolerance and grain length), a sorghum gene that encodes a putative member of the DUF1645 protein family of unknown function. Expression of SbSGL in rice promoted cell division and grain filling, which affected an array of traits of rice, including grain length, grain weight, and seed setting rate. Expression of SbSGL also affected the expression of genes related to the plant cell cycle and grain size.     

The Peptide-Mediated Interactions Between Human Osteoclast-Stimulating Factor and Its Partner Proteins in Osteoporosis: Which Binds to Which?

Human osteoclast-stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption in osteoporosis by recruiting multiple signaling complexes with its diverse biological partners through peptide-mediated interactions (PMIs). The protein contains a modular peptide-recognition domain of Src homology 3 (SH3), which can recognize and bind to the polyproline regions of its partner proteins, as well as two N-terminal polyproline segments, which can be recognized and bound by the SH3 domains of its partner proteins. Here, we attempted to elucidate the complicated PMIs between the different SH3 domains and different polyprolines of hOSF and its three known interacting partners, i.e. proto-oncogene tyrosine-protein kinase (c-Src), survival motor neuron (SMN) and Src-associated in mitosis, 68 kD (Sam68). A total of 29 peptide segments containing the SH3-binding motif PXXP were extracted from these partner proteins, which are potential binding sites of hOSF SH3 domain, while the c-Src kinase also possesses a SH3 domain that may recognize and bind the two polyproline peptides at hOSF N-terminus. Structural bioinformatics analysis identified a number of biologically functional PMI candidates between these SH3 domains and these polyproline peptides, which were then tested in vitro using fluorescence spectroscopy assays. Consequently, it is found that (i) hOSF SH3 domain exhibits strong binding potency to two Sam68 peptides ³⁶RQPPLPHR⁴³ (K _d = 13.7 μM) and ⁴²⁵APPARPVK⁴³² (K _d = 3.2 μM) as well as moderate affinity to three SMN peptides ¹⁹³FLPPPPPM²⁰⁰ (K _d = 56.2 μM), ²³⁵PFPSGPPI²⁴² (K _d = 28.4 μM) and ²⁴⁶PPPICPDS²⁵³ (K _d = 74.5 μM), but has only weak or no binding to c-Src peptides. Instead, a proline-rich region at hOSF N-terminal that contains two overlapping peptides (³KPPPKPVK¹⁰ and ⁶PKPVKPGQ¹³) can be bound tightly by c-Src SH3 domain with high and moderate affinity (K _d = 5.8 and 39.6 μM, respectively).     

Biotechnological potential of a rhizosphere <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.     

Towards the mode of action of <Emphasis Type="Italic">Strobilanthes crispus</Emphasis> through integrated computational and experimental analyses

Bioactive sub-fractions from the tropical herbal plant Strobilanthes crispus (S. crispus) has been shown to induce apoptosis of breast cancer cells in vitro and reduce tumor size in vivo by our earlier studies. We have recently isolated five major compounds from S. crispus sub-fraction, namely lutein, β-sitosterol, campesterol, stigmasterol and pheophytin a. In this study, we set out to investigate each compound’s protein targets and mechanism of action through prediction of protein targets via a ligand-based target prediction protocol, Prediction IncluDinG INactives, and radioligand binding assays. The three phytosterol molecules (β-sitosterol, campesterol, stigmasterol) showed enrichment of hormone signaling GO terms [average ratio (AR) <0.01], while the SMAD signaling pathway was associated with pheophytin a (AR < 0.01). GO terms associated with retinoic acid receptor (RAR) and retinoid X receptor (RXR) were distinctly represented by protein targets of lutein (AR < 0.01). All members of the RAR/RXR family of proteins were predicted to be targeted by lutein, a feature that was not present in the other four S. crispus-derived compounds. Radioligand binding assay in vitro validated that lutein showed higher binding affinity with RXRα (IC₅₀: 5.74 µM; K_i: 4.55 µM) than RARα (IC₅₀: 25.1 µM; K_i: 14 µM). Molecular docking analysis demonstrated that lutein could occupy a large hydrophobic cavity of the hRXRα-LBP crystal structure mainly through hydrophobic interactions with leucine and isoleucine residues, and also hydrogen bond between a hydroxyl group of lutein with Glu²³⁹. Our findings suggest that lutein-RXRα interaction might play a role in the anti-breast cancer effects rendered by S. crispus.     

Elemental composition and flavonol content of <Emphasis Type="Italic">Pentaphylloides fruticosa</Emphasis> (L.) O. Schwarz in connection with plant age

The elemental composition and flavonol content of Pentaphylloides fruticosa (L.) O. Schwartz under the conditions of the Altai mountains are investigated. Some differences in the concentrations of elements and flavonols were detected in the leaves of P. fruticosa plants of different age classes corresponding to two periods of development: pregenerative (immature and virginal plants) and generative (young, middle-aged and old generative individuals). The maximum content of flavonols, in particular quercetin, in the leaves of P. fruticosa corresponds to the young generative age. A very high correlation between the flavonol content and the concentrations of Mn, Zn, Mo, and Se was revealed.     

In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.     

Global insight into the distribution of velvet-like B protein in <Emphasis Type="Italic">Cochliobolus</Emphasis> species and implication in pathogenicity and fungicide resistance

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10 billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.     

Genome-wide identification and characterization of the RIO atypical kinase family in plants

Members of the right open reading frame (RIO) atypical kinase family are present in all three domains of life. In eukaryotes, three subfamilies have been identified: RIO1, RIO2, and RIO3. Studies have shown that the yeast and human RIO1 and RIO2 kinases are essential for the biogenesis of small ribosomal subunits. Thus far, RIO3 has been found only in multicellular eukaryotes. In this study, we systematically identified members of the RIO gene family in 37 species representing the major evolutionary lineages in Viridiplantae. A total of 84 RIO genes were identified; among them, 41 were classified as RIO1 and 43 as RIO2. However, no RIO3 gene was found in any of the species examined. Phylogenetic trees constructed for plant RIO1 and RIO2 proteins were generally congruent with the species phylogeny. Subcellular localization analyses showed that the plant RIO proteins were localized mainly in the nucleus and/or cytoplasm. Expression profile analysis of rice, maize, and Arabidopsis RIO genes in different tissues revealed similar expression patterns between RIO1 and RIO2 genes, and their expression levels were high in certain tissues. In addition, the expressions of plant RIO genes were regulated by two drugs: mycophenolic acid and actinomycin D. Function prediction using genome-wide coexpression analysis revealed that most plant RIO genes may be involved in ribosome biogenesis. Our results will be useful for the evolutionary analysis of the ancient RIO kinase family and provide a basis for further functional characterization of RIO genes in plants.     

Studies in the taxonomy of the Polypores II

The following genera are redefined:Albatrellus S. F. Gray,Heterobasidion Bref.,Haploporus Bond. et Sing. ex Sing.,Fomitopsis P. Karst. andRigidoporus Murrill two new subgenera are described:Polyporus subgen.Dendropolyporus Pouz. (type:Polyporus umbellatus) andRigidoporus subgen.Neooxyporus Pouz. (type:Polyporus latemarginatus); the genusOxyporus (Bourd. etGalz.)Donk is classified as a subgenus of the genusRigidoporus,Murrill and the generaBjerkandera P. Karst. andLeptoporus quél. are classified as subgenera of the genusTyromyces P. Karst. The new subfamilyAlbatrelloideae Pouz. (genera:Albatrellus andGrifola) is described and 14 new specific combinations are made. The new genusIrpicodon Pouz. (type:Irpex pendulus) is proposed.     

Nucleotide Correspondence between Protein-Coding Sequences of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and J99

Comparison of ORFs between H. pylori strains 26695 and J99 showed that transitions (more than 3%) prevail over transversions (less than 1%). The predominance of transitions was explained by the high rates of cytosine replacement by thymine in the coding (3.5–5.3%) and noncoding (2.9–3.9%) DNA strands. The proportion of transversion-type correspondences (A → C, A → T, C → A, C → G, G → C, G → T, T → A, and T → G) did not exceed 0.84%. The highest proportion (28.3%) was observed for correspondences between C and T in ACGT-ATGT, the target site of active methyltransferase of H. pylori J99 (M.Hpy99XI). It was assumed that C → T mutations due to cytosine methylation-deamination are prevalent in H. pylori.     

Alternative methods in toxicology tests: In vitro toxicity

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develope alternative methods to reduce the required number of animals. The newinvitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by theinvitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity withinvitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems forinvitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mytochondrial activity, intracellular content and leakage of enzymes into the medium.     

Flechtensystematische Studien IV. Die GattungGyalidea Lett

Im Artikel wird die ursprüngliche Gattungsdiagnose derGyalidea Lett. um die Merkmale der Pilzkomponente ergänzt. Die angenommenen verwandtschaftlichen Beziehungen derGyalidea zu der FamilieAsterothyriaceae werden diskutiert. Alle bisher festgestellten Arten mit Übersichten der revidierten Proben werden kurz beschrieben. Neue Taxa und Kombinationen:Gyalidea dodgei spec. n.,G. epiphylla spec. n.,G. mayaguezensis spec. n.,G. portoricensis spec. n.,G. fritzei (Stein) comb. n.,G. fritzei var.rivularis (Eitn.) comb. n.,G. hyalinescens (Nyl.) comb. n.,G. lecideopsis var.convarians (Nyl.) comb. n.,G. lecideopsis var.stigmatoides (Nyl.) comb. n.,G. mexicana (B. de Lesd.) comb. n. undG. subscutellaris (Vězda) comb. n.     

Differentiation of species in Gonioloboceras

The type specimen ofGonioloboceras goniolobum (Meek), rediscovered by Spath in the British Museum, is the foundation for a more accurate comparative study of this and other species ofGonioloboceras.Gonioloboceras described asG. goniolobum byElias in 1938 is differentiated asGonioloboceras schmidti, new species. Suture sets (new term) for several growth stages inG. goniolobum (Meek),G. welleriSmith,G. schmidtiElias, G.eliasiMiller &Owen, andG. asiaticumLibrovitch are assembled and used for differentiation of the species.The Kazakhstan goniatite faunule containingG. asiaticum is considered of very late Pennsylvanian age.     

Differences in specificity and compensatory functions among three major starch synthases determine the structure of amylopectin in rice endosperm

Key message

This work adds a new player, HER2, downstream of the perception of E-2-hexenal, a green leaf volatile, and shows that E-2-hexenal specifically changes the redox status of the mitochondria.

Abstract

It is widely accepted that plants produce and respond to green leaf volatiles (GLVs), but the molecular components involved in transducing their perception are largely unknown. The GLV E-2-hexenal inhibits root elongation in seedlings and, using this phenotype, we isolated E-2-hexenal response (her) Arabidopsis thaliana mutants. Using map-based cloning we positioned the her2 mutation to the At5g63620 locus, resulting in a phenylalanine instead of serine on position 223. Knockdown and overexpression lines of HER2 confirmed the role of HER2, which encodes an oxidoreductase, in the responsiveness to E-2-hexenal. Since E-2-hexenal is a reactive electrophile species, which are known to influence the redox status of cells, we utilized redox sensitive GFP2 (roGFP2) to determine the redox status of E-2-hexenal-treated root cells. Since the signal peptide of HER2 directed mCherry to the mitochondria, we targeted the expression of roGFP2 to this organelle besides the cytosol. E-2-hexenal specifically induced a change in the redox status in the mitochondria. We did not see a difference in the redox status in her2 compared to wild-type Arabidopsis. Still, the mitochondrial redox status did not change with Z-3-hexenol, another abundant GLV. These results indicate that HER2 is involved in transducing the perception of E-2-hexenal, which changes the redox status of the mitochondria.

     

Some chromosome numbers of the CzechoslovakLeguminosae

Chromosome numbers of the Czechoslovak species of the genusLotus, from various localities have been determined. The paper includes the speciesLotus uliginosus Schkuhr,L.tenuis Waldst. etKit. andL. borbásii Ujhelyi.     

Heterologous expression of a β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase in <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.