QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the epithelial chloride channel CF transmembrane conductance regulator (CFTR) protein. The most common mutation is a deletion of three nucleotides leading to the loss of phenylalanine at position 508 (p.Phe508del) in the protein. This study evaluates eluforsen, a novel, single-stranded, 33-nucleotide antisense oligonucleotide designed to restore CFTR function, in in vitro and in vivo models of p.Phe508del CF. The aims of the study were to demonstrate cellular uptake of eluforsen, and its efficacy in functional restoration of p.Phe508del-CFTR both in vitro and in vivo. In vitro, the effect of eluforsen was investigated in human CF pancreatic adenocarcinoma cells and human bronchial epithelial cells. Two mouse models were used to evaluate eluforsen in vivo. In vitro, eluforsen improved chloride efflux in CF pancreatic adenocarcinoma cell cultures and increased short-circuit current in primary human bronchial epithelial cells, both indicating restoration of CFTR function. In vivo, eluforsen was taken up by airway epithelium following oro-tracheal administration in mice, resulting in systemic exposure of eluforsen. In female F508del-CFTR mice, eluforsen significantly increased CFTR-mediated saliva secretion (used as a measure of CFTR function, equivalent to the sweat test in humans). Similarly, intranasal administration of eluforsen significantly improved nasal potential difference (NPD), and therefore CFTR conductance, in two CF mouse models. These findings indicate that eluforsen improved CFTR function in cell and animal models of p.Phe508del-CFTR-mediated CF and supported further development of eluforsen in human clinical trials, where eluforsen has also been shown to improve CFTR activity as measured by NPD.

Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the ß-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100.

BACKGROUND: Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds. RESULTS: Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA. CONCLUSION: Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

Modulation of ethylene- and heat-controlled hyponastic leaf movement in Arabidopsis thaliana by the plant defence hormones jasmonate and salicylate.

Upward leaf movement (hyponastic growth) is adopted by several plant species including Arabidopsis thaliana, as a mechanism to escape adverse growth conditions. Among the signals that trigger hyponastic growth are, the gaseous hormone ethylene, low light intensities, and supra-optimal temperatures (heat). Recent studies indicated that the defence-related phytohormones jasmonic acid (JA) and salicylic acid (SA) synthesized by the plant upon biotic infestation repress low light-induced hyponastic growth. The hyponastic growth response induced by high temperature (heat) treatment and upon application of the gaseous hormone ethylene is highly similar to the response induced by low light. To test if these environmental signals induce hyponastic growth via parallel pathways or converge downstream, we studied here the roles of Methyl-JA (MeJA) and SA on ethylene- and heat-induced hyponastic growth. For this, we used a time-lapse camera setup. Our study includes pharmacological application of MeJA and SA and biological infestation using the JA-inducing caterpillar Pieris rapae as well as mutants lacking JA or SA signalling components. The data demonstrate that MeJA is a positive, and SA, a negative regulator of ethylene-induced hyponastic growth and that both hormones repress the response to heat. Taking previous studies into account, we conclude that SA is the first among many tested components which is repressing hyponastic growth under all tested inductive environmental stimuli. However, since MeJA is a positive regulator of ethylene-induced hyponastic growth and is inhibiting low light- and heat-induced leaf movement, we conclude that defence hormones control hyponastic growth by affecting stimulus-specific signalling pathways.

In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs.

ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.

Apolipoprotein B knockdown by AAV-delivered shRNA lowers plasma cholesterol in mice.

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.

Kinome profiling reveals an interaction between jasmonate, salicylate and light control of hyponastic petiole growth in Arabidopsis thaliana.

Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ∼omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.

Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway.

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.

Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic Acid.

Cross-talk between jasmonate (JA), ethylene (ET), and Salicylic acid (SA) signaling is thought to operate as a mechanism to fine-tune induced defenses that are activated in response to multiple attackers. Here, 43 Arabidopsis genotypes impaired in hormone signaling or defense-related processes were screened for their ability to express SA-mediated suppression of JA-responsive gene expression. Mutant cev1, which displays constitutive expression of JA and ET responses, appeared to be insensitive to SA-mediated suppression of the JA-responsive marker genes PDF1.2 and VSP2. Accordingly, strong activation of JA and ET responses by the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola prior to SA treatment counteracted the ability of SA to suppress the JA response. Pharmacological assays, mutant analysis, and studies with the ET-signaling inhibitor 1-methylcyclopropene revealed that ET signaling renders the JA response insensitive to subsequent suppression by SA. The APETALA2/ETHYLENE RESPONSE FACTOR transcription factor ORA59, which regulates JA/ET-responsive genes such as PDF1.2, emerged as a potential mediator in this process. Collectively, our results point to a model in which simultaneous induction of the JA and ET pathway renders the plant insensitive to future SA-mediated suppression of JA-dependent defenses, which may prioritize the JA/ET pathway over the SA pathway during multi-attacker interactions.

Are small GTPases signal hubs in sugar-mediated induction of fructan biosynthesis?

External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling.

Kinome profiling using peptide arrays in eukaryotic cells.

Over the last 10 years array and mass spectrometry technologies have enabled the determination of the transcriptome and proteome of biological and in particular eukaryotic systems. This information will likely be of significant value to our elucidation of the molecular mechanisms that govern eukaryotic physiology. However, an equally, if not more important goal, is to define those proteins that participate in signalling pathways that ultimately control cell fate. Enzymes that phosphorylate tyrosine, serine, and threonine residues on other proteins play a major role in signalling cascades that determine cell-cycle entry, and survival and differentiation fate in the tissues across the eukaryotic kingdoms. Knowing which signalling pathways are being used in these cells is of critical importance. Traditional genetic and biochemical approaches can certainly provide answers here, but for technical and practical reasons there is typically pursued one gene or pathway at a time. Thus, a more comprehensive approach is needed in order to reveal signalling pathways active in nucleated cells. Towards this end, kinome analysis techniques using peptide arrays have begun to be applied with substantial success in a variety of organisms from all major branches of eukaryotic life, generating descriptions of cellular signalling without a priori assumptions as to possibly effected pathways. The general procedure and analysis methods are very similar disregarding whether the primary source of the material is animal, plant, or fungal of nature and will be described in this chapter. These studies will help us better understand what signalling pathways are critical to controlling eukaryotic cell function.

Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling.

The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play crucial roles in the signaling network that regulates induced defense responses against biotic stresses. Antagonism between SA and JA operates as a mechanism to fine-tune defenses that are activated in response to multiple attackers. In Arabidopsis (Arabidopsis thaliana), NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) was demonstrated to be required for SA-mediated suppression of JA-dependent defenses. Because ET is known to enhance SA/NPR1-dependent defense responses, we investigated the role of ET in the SA-JA signal interaction. Pharmacological experiments with gaseous ET and the ET precursor 1-aminocyclopropane-1-carboxylic acid showed that ET potentiated SA/NPR1-dependent PATHOGENESIS-RELATED1 transcription, while it rendered the antagonistic effect of SA on methyl jasmonate-induced PDF1.2 and VSP2 expression NPR1 independent. This overriding effect of ET on NPR1 function in SA-JA cross talk was absent in the npr1-1/ein2-1 double mutant, demonstrating that it is mediated via ET signaling. Abiotic and biotic induction of the ET response similarly abolished the NPR1 dependency of the SA-JA signal interaction. Furthermore, JA-dependent resistance against biotic attackers was antagonized by SA in an NPR1-dependent fashion only when the plant-attacker combination did not result in the production of high levels of endogenous ET. Hence, the interaction between ET and NPR1 plays an important modulating role in the fine tuning of the defense signaling network that is activated upon pathogen and insect attack. Our results suggest a model in which ET modulates the NPR1 dependency of SA-JA antagonism, possibly to compensate for enhanced allocation of NPR1 to function in SA-dependent activation of PR genes.

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase.

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.

Kinome profiling of sugar signaling in plants using multiple platforms.

Although the primary sequence of kinases shows substantial divergence between unrelated eukaryotes, variation in the motifs that are actually phosphorylated by eukaryotic kinases is much smaller. Hence arrays developed for kinome profiling of mammalian cells are useful for kinome profiling of plant tissues as well, facilitating the study of plant signal transduction. We recently employed the Pepscan kinomics chip to reveal the small GTPases in plant sucrose signaling. Here we show that employing a different peptide library (the Pepscan kinase chip) largely similar results are obtained, confirming these earlier data, but such a different library also contributes new insights into the molecular details mediating plant cell responses to a sugar stimulus. Thus when studying plant signal transduction employing peptide arrays, using multiple platforms both increases the confidence of results and provides additional information.

Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation.

Cross talk between salicylic acid (SA) and jasmonic acid (JA) signaling pathways plays an important role in the regulation and fine tuning of induced defenses that are activated upon pathogen or insect attack. Pharmacological experiments revealed that transcription of JA-responsive marker genes, such as PDF1.2 and VSP2, is highly sensitive to suppression by SA. This antagonistic effect of SA on JA signaling was also observed when the JA pathway was biologically activated by necrotrophic pathogens or insect herbivores, and when the SA pathway was triggered by a biotrophic pathogen. Furthermore, all 18 Arabidopsis (Arabidopsis thaliana) accessions tested displayed SA-mediated suppression of JA-responsive gene expression, highlighting the potential significance of this phenomenon in induced plant defenses in nature. During plant-attacker interactions, the kinetics of SA and JA signaling are highly dynamic. Mimicking this dynamic response by applying SA and methyl jasmonate (MeJA) at different concentrations and time intervals revealed that PDF1.2 transcription is readily suppressed when the SA response was activated at or after the onset of the JA response, and that this SA-JA antagonism is long lasting. However, when SA was applied more than 30 h prior to the onset of the JA response, the suppressive effect of SA was completely absent. The window of opportunity of SA to suppress MeJA-induced PDF1.2 transcription coincided with a transient increase in glutathione levels. The glutathione biosynthesis inhibitor l-buthionine-sulfoximine strongly reduced PDF1.2 suppression by SA, suggesting that SA-mediated redox modulation plays an important role in the SA-mediated attenuation of the JA signaling pathway.

Evidence for a minimal eukaryotic phosphoproteome?

BACKGROUND: Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in cell lysates from representatives of various branches of the eukaryotic species. We derived a set of substrates from the PhosphoBase whose phosphorylation by cellular extracts is common to the divergent members of different kingdoms and thus may be considered a minimal eukaryotic phosphoproteome. The protein kinases (or kinome) responsible for phosphorylation of these substrates are involved in a variety of processes such as transcription, translation, and cytoskeletal reorganisation. CONCLUSIONS/SIGNIFICANCE: These results indicate that the divergence in eukaryotic kinases is not reflected at the level of substrate phosphorylation, revealing the presence of a limited common substrate space for kinases in eukaryotes and suggests the presence of a set of kinase substrates and regulatory mechanisms in an ancestral eukaryote that has since remained constant in eukaryotic life.

Kinome profiling of Arabidopsis using arrays of kinase consensus substrates.

BACKGROUND: Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. RESULTS: Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities. Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. CONCLUSION: We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown) kinases that play a role in P. syringae infection. This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.

Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.

Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.

Using natural variation to investigate the function of individual amino acids in the sucrose-binding box of fructan:fructan 6G-fructosyltransferase (6G-FFT) in product formation.

Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema et al., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the beta-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87.