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S Smeekens

Publications and source records attributed to S Smeekens.

At least 19 recordsLinked to original sources
PubMed2005

Expression and characterization of a serine protease that preferentially cleaves insulin-like growth factor binding protein-5.

Insulin-like growth factor binding proteins (IGFBPs) play important roles in regulating the functions of insulin-like growth factors (IGFs). Because IGFBPs have very high affinity for IGF-I and IGF-II, they can regulate the amount of each growth factor that is able to bind to cell surface receptors, therefore, factors that alter IGFBP affinity have the capacity to regulate IGF actions. Protease activities that are present in cell culture systems and physiologic fluids have been shown to degrade IGFBP-5. Previously, a region of sequence in a serine protease was identified that was homologous with the N-terminal 90 amino acids of members of the IGFBP family and with members of the CCN family of proteins. In a prior study, the protease was expressed in human kidney cultured cells and the cell culture supernatants were shown to cleave IGFBP-5, however, it is unknown whether the purified protease would cleave IGFBP-5 and whether it would also cleave other specific forms of IGFBPs. In this study, we expressed this protease in an insect cell expression system, purified it to homogeneity and tested its capacity to cleave IGFBP-5. The expressed protease preferentially cleaved IGFBP-5, and it had minimal activity toward other forms of IGFBPs. The proteolytic activity of this IGFBPase is inhibited by serine protease inhibitors including PMSF and 3,4-dichloroisocoumarin, as well as by divalent metal ions such as, Zn and Cu. Mutation of the active site serine resulted in a major reduction in IGFBP-5 cleavage. The protease binds to heparin and its ability to degrade IGFBP-5 is blocked in the presence of heparin. Inhibition of the activity of the protease following its secretion by B104 cells resulted in inhibition of IGFBP-5 proteolysis and IGF-I stimulation of protein synthesis. Northern blotting revealed that the transcript was expressed in multiple human tissues, including placenta, uterus, prostate, testis, spinal cord, brain, liver, small intestine, thyroid, and spleen. The highest expression was in uterus and placenta, suggesting a possible role of sex steroids in regulating its expression. Understanding the mechanism of how cleavage of IGFBP-5 by this protease alters its activity will help to further our understanding of the biologic actions of the IGFs.

Base Sequence↗
PubMed2005

Sucrose-induced translational repression of plant bZIP-type transcription factors.

Sugars as signalling molecules exert control on the transcription of many plant genes. Sugar signals also alter mRNA and protein stability. Increased sucrose concentrations specifically repress translation of the S-class basic region leucine zipper (bZIP) type transcription factor AtbZIP11/ATB2. This sucrose-induced repression of translation (SIRT) depends on translation of a highly conserved upstream open reading frame (uORF) in the 5' UTR of the gene. This conserved uORF is exclusively encoded in 5' UTRs of several plant S-class bZIP transcription factors. Arabidopsis homologues of ATB2/AtbZIP11, which harbour the conserved uORF, also show SIRT. Therefore, SIRT emerges as a general sucrose translational control mechanism of a group of transcription factors. SIRT might be part of a sucrose-specific signalling pathway, controlling expression of plant bZIP transcription factor genes.

Open Reading Frames↗
PubMed2000

The Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses.

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled-6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase-dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE-4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase-dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid- and sugar-dependent way.

Abscisic Acid↗
PubMed1999

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Arabidopsis↗
PubMed1998

Sugar regulation of gene expression in plants.

The molecular details of sugar sensing and sugar-mediated signal transduction pathways are unclear but recent results suggest that hexokinase functions as an important plant sugar sensor in a way that is similar to that found in yeast. The use of mutants in Arabidopsis defective in specific signaling steps is of particular importance because these give access to the genes encoding components in the signaling pathways. In addition, the physiological analysis of such mutants may reveal the interaction of sugar-induced signaling pathways and those induced by other stimuli such as environmental or biotic stress.

Gene Expression Regulation, Plant↗
PubMed1998

The light-regulated Arabidopsis bZIP transcription factor gene ATB2 encodes a protein with an unusually long leucine zipper domain.

A light-regulated basic domain/leucine zipper gene, ATB2, was identified in an Arabidopsis thaliana transcription factor gene collection. Both genomic and cDNA clones of ATB2 were isolated. The gene encodes a small protein (18 kDa) which mainly consists of the basic domain and an unusually long leucine zipper. The expression of the ATB2 gene is induced when etiolated or dark-adapted seedlings are transferred to the light. Moreover, its expression is derepressed in dark-grown seedlings of the photomorphogenic mutants cop1 and det1. In mature plants, transcript levels are particularly high in flowers and also light-responsive in these tissues.

Adaptation, Biological↗
PubMed1998

Sensing trehalose biosynthesis in plants.

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.

Genes, Plant↗
PubMed1998

Sucrose-specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene.

The Arabidopsis bZIP transcription factor gene ATB2 has been shown previously to be expressed in a light-regulated and tissue-specific way. Here we describe the precise localization of ATB2 expression, using transgenic lines containing an ATB2 promoter-GUS reporter gene construct. The observed expression pattern suggests a role for ATB2 in the control of processes associated with the transport or utilization of metabolites. Remarkably, expression of the ATB2-GUS reporter gene construct was specifically repressed by sucrose. Other sugars, such as glucose and fructose, alone or in combination, were ineffective. Repression was observed at external sucrose concentrations exceeding 25 mM. Transcript levels of both the endogenous ATB2 gene and the ATB2-GUS reporter gene were not repressed by sucrose, suggesting that sucrose affects mRNA translation. This translational regulation involves the ATB2 leader sequence because deletion of the leader resulted in loss of sucrose repression. Our results provide evidence for a sucrose-specific sugar sensing and signalling system in plants.

Arabidopsis↗
PubMed1998

Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana.

Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

Amino Acid Sequence↗
PubMed1998

Cloning of sucrose:sucrose 1-fructosyltransferase from onion and synthesis of structurally defined fructan molecules from sucrose.

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Amino Acid Sequence↗
PubMed1997

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase.

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.

Allium↗
PubMed1996

A tobacco nuclear protein that preferentially binds to unmethylated CpG-rich DNA.

The methylation of cytosine residues in CpG dinucleotides of eukaryotic DNA is an important mechanism for the regulation of gene expression. Higher plants have a high content of methylated cytosine residues in CpG as well as CpNpG sites, and experimental evidence suggests a role in gene expression for DNA methylation. In this article, we describe a tobacco nuclear protein whose binding to various DNA sequences is positively correlated with the CpG density of the probes. This protein, CpG-binding protein 1 (CGBP-1), has reduced affinity for DNA when the CpG sites are methylated. Ribonuclease treatment also reduces the formation of the CGBP-1 complex. The binding characteristics of CGBP-1 make it an interesting protein with respect to methylation-mediated gene expression in plants.

Base Sequence↗
PubMed1996

Identification of a light-regulated MYB gene from an Arabidopsis transcription factor gene collection.

Seven different MYB-related genes have been isolated from a genomic Arabidopsis library with probes based on MYB DNA-binding motifs. The predicted amino acid sequence of these genes showed high similarity in the MYB domain but outside this region virtually no similarities were found. The set of MYB-related genes was used to identify differentially expressed genes following the transfer of etiolated seedlings to light. This differential screen resulted in the selection of the ATM4 gene which is induced by light within one hour of exposure of etiolated or dark-adapted seedlings.

Amino Acid Sequence↗
PubMed1995

Light-regulated expression of the Arabidopsis thaliana ferredoxin gene requires sequences upstream and downstream of the transcription initiation site.

The effect of light on the expression of the Arabidopsis thaliana ferredoxin gene (fedA) was studied in mature tobacco plants. In light-treated leaves of tobacco plants transformed with a full-length ferredoxin gene, fedA-specific mRNA levels were more than twenty fold higher than in dark-treated controls. This indicates that all components for regulation of the Arabidopsis ferredoxin gene are present in tobacco. To identify light-regulatory elements in the fedA gene, we have tested a set of chimeric genes containing various parts of the fedA gene for light-dependent expression in mature tobacco plants. A fedA promoter-GUS fusion gene was not light-responsive, indicating that the 5'-upstream promoter region is not sufficient for light regulation. Fusion genes in which different transcribed regions of the fedA gene were expressed from the CaMV 35S promoter showed only limited light regulation, if any at all. This indicates that, like the fedA upstream region, the region downstream of the transcription start site is also not sufficient for full light regulation. The combined results suggest that for full light-regulated expression of the fedA gene, both the promoter region and sequences downstream of the transcription start site are required.

Arabidopsis↗
PubMed1995

The homeobox gene ATK1 of Arabidopsis thaliana is expressed in the shoot apex of the seedling and in flowers and inflorescence stems of mature plants.

The homeodomain is a DNA-binding domain present in a large family of eukaryotic regulatory proteins. Homeodomain proteins have been shown to play key roles in controlling developmental programs in various organisms. Here we report the isolation and characterisation of a homeobox gene from Arabidopsis thaliana designated ATK1. The gene was isolated using as a probe the homeobox domain of the KN1 gene from maize. The homeodomain of ATK1 is highly homologous to the homeodomain of the KN1 gene of maize (81%) but shows only poor homology outside the homeodomain. Therefore ATK1 is probably not the Arabidopsis homologue of the KN1 gene from maize. It contains the four invariant amino acid residues present in the recognition helix 3 of all other homeodomain proteins. Outside the homeodomain a region rich in aspartate and glutamate residues is found suggesting that ATK1 is a transcriptional activator. The gene contains four introns which is similar in the KN1 gene of maize and the Osh1 gene of rice. Primer extension reveals the presence of two transcription initiation sites. The leader sequence of the genuine transcript is 342 nucleotides long and contains two upstream open reading frames. ATK1 is strongly expressed in the shoot apex of seedlings, while in mature plants the gene is primarily expressed in flowers and inflorescence stems. Such an expression pattern is reminiscent of that of the KN1 gene of maize and therefore ATK1 could similarly be involved in determining cell fate.

Amino Acid Sequence↗
PubMed1995

The homeobox gene ATH1 of Arabidopsis is derepressed in the photomorphogenic mutants cop1 and det1.

A light-regulated Arabidopsis thaliana homeobox 1 gene (ATH1) was identified in a transcription factor gene collection. Primer extension analysis of this gene showed the presence of two major transcripts with unusually long 5' untranslated leader sequences. The leader sequence of the isolated cDNA clone contains several small open reading frames upstream of the initiation codon of the largest open reading frame coding for the homeodomain protein. This leader sequence was found to affect the translation efficiency negatively in an in vitro translation system. The expression of the ATH1 gene is dependent on the presence of light. After illuminating etiolated or dark-adapted Arabidopsis seedlings, the ATH1 mRNA level increased rapidly. Expression of ATH1 does not require the presence of active chloroplasts because photooxidative destruction of the chloroplast by norflurazon treatment did not influence the ATH1 mRNA level. In dark-grown seedlings of the photomorphogenic constitutive photomorphogenic 1 (cop1) and deetiolated 1 (det1) mutants, the ATH1 mRNA level was elevated. This shows that the gene products of these loci directly or indirectly repress ATH1 expression in etiolated wild-type seedlings. A correlation between the strength of the cop1 allele and the ATH1 mRNA level was found. This relationship suggests a role for the ATH1 protein in the signal transduction pathway downstream of COP1.

Amino Acid Sequence↗