Funktionelle Analyse der LC-FACS in Dictyostelium discoideum

"Funktionelle Analyse der LC-FACS in Dictyostelium discoideum" Das Dictyostelium discoideum Gen fcsA kodiert für ein 75 kDa großes Protein. Es kann durch Homologieanyalysen der Amino-säuresequenz zu den "long-chain fatty acyl-CoA"-Synthetasen ge-rechnet werden, die lang-kettige Fettsäuren durch die kovalente Bindung von Coenzym A akti-vie-ren und damit für diverse Reak-tionen in Stoffwechsel und Molekül-Synthese der Zelle verfügbar machen. Die hier untersuchte D. discoideum LC-FACS lokalisiert als peripher assoziiertes Protein an der cytosolischen Seite der Membran von Endo-somen und kleiner Vesikel. Bereits kurz nach der Bildung in der frühen sauren Phase kann die Lokalisation der LC-FACS auf Endosomen ge-zeigt werden. Sie dissoziiert im Laufe ihrer Neutra-li-sierung und kann auf späten Endosomen, die vor ihrer Exocytose stehen nicht mehr nach-gewiesen werden. Ein Teil der kleinen die in der gesamte Zelle verteilten kleinen Vesikel zeigt eine Kolokalisation mit lysosomalen Enzymen. Trotz des intrazellulären Verteilungs-mus-ters, das eine Beteiligung dieses Pro-teins an der Endocytose nahe-legt, konnte kein signifikanter Rückgang der Pino- und Phagocytose-Rate in LC-FACS Nullmutanten beobachtet werden. Der endo-cy-to-ti-sche Transit ist in diesen Zellen etwas verlängert, außerdem zeigen die Endosomen einen deutlich erhöhten pH-Wert, was zu einer weniger effektiven Prozessierung eines lysosomalen Enzyms führt (a-Mannosidase). Die Funktion der LC-FACS ist die Aufnahme von langkettigen Fettsäuren aus dem Lumen der Endosomen.

Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.

Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Cupins: the most functionally diverse protein superfamily?

The cupin superfamily of proteins, named on the basis of a conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), was originally discovered using a conserved motif found within germin and germin-like proteins from higher plants. Previous analysis of cupins had identified some 18 different functional classes that range from single-domain bacterial enzymes such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors including one linked to the nodulation response in legumes. Recent advances in comparative genomics, and the resolution of many more 3-D structures have now revealed that the largest subset of the cupin superfamily is the 2-oxyglutarate-Fe2+ dependent dioxygenases. The substrates for this subclass of enzyme are many and varied and in total amount to probably 50–100 different biochemical reactions, including several involved in plant growth and development. Although the majority of enzymatic cupins contain iron as an active site metal, other members contain either copper, zinc, cobalt, nickel or manganese ions as a cofactor, with each cofactor allowing a different type of chemistry to occur within the conserved tertiary structure. This review discusses the range of structures and functions found in this most diverse of superfamilies.

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two-dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI - TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two-dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin-like proteins or glucanases, and cell expansion, i.e. β-D-glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.

Gene expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the potential for diagnostic gene expression markers

Background: There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples. Results: We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes. Conclusions: This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: alpha-helix (20%), beta-sheet (33%), turned (19%) and unordered (28%). RMSD 1%. CpOsm was stable at up to 75 degrees C, and thermal denaturation (T(m)) was calculated to be 77.8 degrees C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins. (C) 2011 Elsevier B.V. All rights reserved.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected rootstock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Downregulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [³⁵S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

Field evaluation of a suite of biomarkers in an Australian Tropical Reef Species, Stripey Seaperch (Lutjanus carponotatus) : Assessment of Produced Formation water from the Harriet A Platform

There is paucity of data regarding hydrocarbon exposure of tropical fish species inhabiting the waters near oil and gas platforms on the Northwest Shelf of Australia. A comprehensive field study assessed the exposure and potential effects associated with the produced water (PW) plume from the Harriet A production platform on the northwest shelf in a local reef species, Stripey seaperch (Lutjanus carponotatus). This field study was a continuation of an earlier pilot study which concluded that there were “warning signs” of potential biological effects on fish populations exposed to PW. A 10-day field caging study was conducted deploying 15 individual fish into 6 separate steel cages set 1-m subsurface at 3 stations in a concentration gradient moving away from the platform. A battery of biomarkers were evaluated including hepatosomatic index (HSI), total cytochrome P450, bile metabolites, CYP1A-, CYP2K- and CYP2M-like proteins, cholinesterase (ChE) activity, and histopathology of liver and gill tissues. Water column and PW effluent samples was also collected. Results confirmed that PAH metabolites in bile, CYP1A-, CYP2K-, and CYP2M-like proteins and liver histopathology provided evidence of significant exposure and effects after 10 days at the near-field site (~200 m off the Harriet A platform). Hepatosomatic index, total cytochrome P450, and ChE did not provide site-specific differences by day 10 of exposure to PW. CYP proteins were shown by principal component analysis (PCA) to be the best diagnostic tool for determining exposure and associated biological effects of PW on L. carponotatus. Using a suite of biomarkers has been widely advocated as a vital component in environmental risk assessments worldwide. This study demonstrates the usefulness of biomarkers for assessing the Harriet A PW discharge into Australian waters with broader applications for other PW discharges. This approach has merit as a valuable addition to environmental management strategies for protecting Australia’s tropical environment and its rich biodiversity.

Structure-function relationship of new crotamine isoform from the Crotalus durissus cascavella

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7-3.0-fold than to control. The Na+ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na+ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic beta-cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.