María J. Lafuente

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae

We have determined that the mutant genes DGT1‐1 and BPC1‐1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two‐hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.

Genetic inactivation of Par4 results in hyperactivation of NF-κB and impairment of JNK and p38

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF‐κB pathway. This may explain its pro‐apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4−/− mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor‐α (TNF‐α) to induce apoptosis by increased activation of NF‐κB. Consistent with recent reports demonstrating the antagonistic actions of NF‐κB and c‐Jun amino‐terminal kinase (JNK) signalling, we have found that Par4−/− cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF‐α and interleukin 1. Higher levels of an anti‐apoptotic JNK‐inhibitor protein, X‐chromosome‐linked inhibitor of apoptosis, in Par4−/− EFs might explain the inhibition of JNK activation in these cells.

Regulation of mature T lymphocyte proliferation and differentiation by Par-4.

The genetic inactivation of the atypical protein kinase C (aPKC) inhibitor, Par‐4, gives rise to increased NF‐κB activation and decreased stimulation of JNK in embryo fibroblasts. Here we have characterized the immunological phenotype of the Par‐4−/− mice and found that the loss of this gene leads to an increased proliferative response of peripheral T cells when challenged through the TCR. This is accompanied by a higher increase in cell cycle entry and inhibition of apoptosis, with enhanced IL‐2 secretion but normal CD25 synthesis. Interestingly, the TCR‐triggered activation of NF‐κB was augmented and that of JNK was severely abrogated. Consistent with previous data from knock outs of different JNKs, NFATc1 activation and IL‐4 secretion were augmented in the Par‐4‐deficient CD4+ T cells, suggesting that the loss of Par‐4 drives T‐cell differentiation towards a Th2 response. This is compelling evidence that Par‐4 is a novel modulator of the immune response through its ability to impact aPKC activity, which translates into lower JNK signaling.

A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe

We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli. These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ. The plasmids were constructed with the ura4+ or the his3+ marker of S. pombe. Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6‐bisphosphatase and β‐galactosidase under the control of the fbp1+ promoter in different conditions.

Case Study of Small Molecules As Antimalarials: 2-Amino-1-phenylethanol (APE) Derivatives.

Antiparasitic oral drugs have been associated to lipophilic molecules due to their intrinsic permeability. However, these kind of molecules are associated to numerous adverse effects, which have been extensively studied. Within the Tres Cantos Antimalarial Set (TCAMS) we have identified two small, soluble and simple hits that even presenting antiplasmodial activities in the range of 0.4-0.5 μM are able to show in vivo activity.

Adaptive Correction from Virtually Complex Dynamic Libraries: The Role of Noncovalent Interactions in Structural Selection and Folding.

The hierarchical self-assembling of complex molecular systems is dictated by the chemical and structural information stored in their components. This information can be expressed through an adaptive process that determines the structurally fittest assembly under given environmental conditions. We have set up complex disulfide-based dynamic covalent libraries of chemically and topologically diverse pseudopeptidic compounds. We show how the reaction evolves from very complex mixtures at short reaction times to the almost exclusive formation of a major compound, through the establishment of intramolecular noncovalent interactions. Our experiments demonstrate that the systems evolve through error-check and error-correction processes. The nature of these interactions, the importance of the folding and the effects of the environment are also discussed.

Disruption and basic functional analysis of six novel ORFs of chromosome XV from Saccharomyces cerevisiae

We report the disruption and functional analysis of six open reading frames (ORFs) from chromosome XV, namely YOL155c, YOL154w, YOL119c, YOL118c, YOR301w and YOR306c, in FY1679 and CEN.PK2 backgrounds. We constructed replacement cassettes and cloned each ORF into the pRS416 centromeric plasmid. No obvious phenotype was observed for the corresponding deleted strains with respect to growth, mating or sporulation. YOL155c encodes a protein with a secretion signal and putative GPI‐anchor recognition site and is possibly a cell wall protein, although its deletion did not present morphogenetic defects under any of the conditions tested. Although YOL119c and YOR306c are members of the monocarboxylate permease family, the growth of the double disruptant in acetate, lactate and pyruvate was similar to that of the parental strains. Copyright

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

Analysis of the DNA Sequence of a 15,500 bp Fragment near the Left Telomere of Chromosome XV from Saccharomyces cerevisiae Reveals a Putative Sugar Transporter, a Carboxypeptidase Homologue and Two New Open Reading Frames

We report the sequence of a 15·5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence. The nucleotide sequence has been deposited in the EMBL data library under Accession Number X89715.

DNA sequence analysis of a 10 624 bp fragment of the left arm of chromosome XV from Saccharomyces cerevisiae reveals a RNA binding protein, a mitochondrial protein, two ribosomal proteins and two new open reading frames

We have determined the sequence of a 10 624 bp DNA segment located in the left arm of chromosome XV of Saccharomyces cerevisiae. The sequence contains eight open reading frames (ORFs) longer than 100 amino acids. Two of them do not present significant homology with sequences found in the databases. The product of ORF o0553 is identical to the protein encoded by the gene SMF1. Internal to it there is another ORF, o0555 that is apparently expressed. The proteins encoded by ORFs o0559 and o0565 are identical to ribosomal proteins S19.e and L18 respectively. ORF o0550 encodes a protein with an RNA binding signature including RNP motifs and stretches rich in asparagine, glutamine and arginine. The nucleotide sequence determined has been deposited in the EMBL data library under Accession Number X95258.