Jan-Paul Bebelman

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects.

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37°C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature‐induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall‐degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature‐induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high‐osmolarity response routes.

Cdc37p Is Required for Stress-Induced High-Osmolarity Glycerol and Protein Kinase C Mitogen-Activated Protein Kinase Pathway Functionality by Interaction with Hog1p and Slt2p (Mpk1p)

ABSTRACT The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.

Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae.

The BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high‐affinity permease for branched‐chain amino acids. In this paper, we show that, like BAP2, the expression of the BAP3 gene in S. cerevisiae is induced by the addition of branched‐chain amino acids to the medium. Unexpectedly, most other naturally occurring L‐amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3. The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre‐tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1 − mutant. The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a Δleu3 mutant. By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from − 418 to − 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p‐dependent induction of BAP3 expression by (most) amino acids. We have therefore named this sequence UASaa (amino acid‐dependent upstream activator sequence). Neither Stp1p nor Leu3p appear to bind to the UASaa, at least in vitro, as judged from gel retardation assays. Sequences similar to the UASaa can be found in the promoters of BAP2, PTR2 and TAT1 ; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.

The CXCR3/CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection

The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system.

Signalling pathways leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast.

Addition of glucose to yeast cells growing on less preferred carbon sources triggers profound changes in the expression levels of several genes. This paper focuses on the signal transduction pathways leading to transcriptional activation of the glycolysis in Saccharomyces cerevisiae during the transition from respiratory to fermentative growth conditions. To this end, we studied the transcriptional regulation of glycolytic genes (PFK1, PYK1 and PDC ), one gluconeogenic gene (FBP1) and the two genes encoding the 6‐phosphofructo‐2‐kinase isoenzymes (PFK26 and PFK27 ) during this transition. The results of experiments using glycolysis mutants, different fermentable carbon sources and 2‐deoxyglucose indicate that proper transcriptional regulation of these genes is dependent on the ability to form glucose 6‐phosphate by any one of the three hexose kinases. In addition, we conclude that signalling via the Ras–adenylate cyclase pathway is not necessary for the proper transcriptional response of glycolytic and gluconeogenic genes to glucose, because the transcription of these genes is not significantly affected in mutants having either high or low activities of this pathway. In contrast, the transcriptional regulation of the PFK26 and PFK27 genes is significantly altered in several of the Ras–adenylate cyclase pathway mutants studied, indicating that protein kinase A plays an important role in the transcriptional regulation of these genes.

Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae.

Changing the growth mode of Saccharomyces cerevisiae by adding fermentable amounts of glucose to cells growing on a non-fermentable carbon source leads to rapid repression of general stress-responsive genes like HSP12. Remarkably, glucose repression of HSP12 appeared to occur even at very low glucose concentrations, down to 0.005%. Although these low levels of glucose do not induce fermentative growth, they do act as a growth signal, since upon addition of glucose to a concentration of 0.02%, growth rate increased and ribosomal protein gene transcription was up-regulated. In an attempt to elucidate how this type of glucose signalling may operate, several signalling mutants were examined. Consistent with the low amounts of glucose that elicit HSP12 repression, neither the main glucose-repression pathway nor cAMP-dependent activation of protein kinase A appeared to play a role in this regulation. Using mutants involved in glucose metabolism, evidence was obtained suggesting that glucose 6-phosphate serves as a signalling molecule. To identify the target for glucose repression on the promoter of the HSP12 gene, a promoter deletion series was used. The major transcription factors governing (stress-induced) transcriptional activation of HSP12 are Msn2p and Msn4p, binding to the general stress-responsive promoter elements (STREs). Surprisingly, glucose repression of HSP12 appeared to be independent of Msn2/4p: HSP12 transcription in glycerol-grown cells was unaffected in a deltamsn2deltamsn4 strain. Nevertheless, evidence was obtained that STRE-mediated transcription is the target of repression by low amounts of glucose. These data suggest that an as yet unidentified factor is involved in STRE-mediated transcriptional regulation of HSP12.

Cloning and sequencing of Rhesus monkey pepsinogen A cDNA

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases.

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.

Transcription Regulation of Human and Porcine Pepsinogen A

The gene regulation of pepsinogens is scientifically interesting, because of the high specificity of expression in gastric chief cells. Also from a clinical point of view pepsinogen gene regulation is interesting. Pepsinogen, activated by acid to pepsin, is one of the aggressive factors in the stomach that play a role in peptic ulcer disease (1). A dominantly inherited high pepsinogen A (PGA) output is associated with duodenal ulcer (2). On the other hand, a low production of pepsinogen A appears to be associated with atrophic gastritis and gastric cancer (3). Changes in gene expression of different isozymogens have been demonstrated in Barrett’s oesophagus (4) and gastric cancer (5). Defize et al. (6) showed, that these changes in gene expression may be associated with methylation of DNA. They demonstrated that N-methyl-N’nitro-N-nitrosoguanidine (MNNG), a methylating agent, caused changes in pepsinogen C (PGC) gene expression in rats and in PGA gene expression in cultured human gastric chief cells. In rats the changes in PGC expression were associated with the development of gastric cancer. Other studies have shown hypomethylation of the pepsinogen genes in pepsinogen producing tissue, i.e. fundic mucosa (7,8). A decrease of PG synthesis in gastric tumours was correlated with a different methylation pattern (9).

Analysis of the Promoter of a Human Pepsinogen a Gene

Pepsinogen A (PGA), inactive precursor of the aspartic proteinase pepsin A (E.C. 3.4.23.1), is encoded on the human genome (chromosome 11ql2-13) by a multigene family1. Electrophoretic separation of the PGA isozymogens from urine or gastric mucosa reveals three main fractions: Pg3, Pg4 and Pg5. The electrophoretic patterns of PGA show a large, genetically-determined, inter-individual heterogeneity. These differences can be explained by the presence of a number of haplotypes, containing different numbers and types of PGA genes2. The study of PGA gene clusters by RFLP analysis previously enabled us to define the haplotypes corresponding to certain phenotypes3. In this paper we describe part of our studies aimed at identifying cis-acting elements and trans-acting factors involved in the tissue-specific transcriptional regulation of PGA gene expression.