Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer

Background

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods

Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Results

Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

Conclusions

As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

General significance

The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.     

In vivo effects of N/OFQ(1–13)NH2 and its structural analogue [ORN9]N/OFQ(1–13)NH2 on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

The effects of nociceptin(1–13)NH₂ (N/OFQ(1–13)NH₂) and its structural analogue [Orn⁹]N/OFQ(1–13)NH₂ on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1–13)NH₂ decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn₉]N/OFQ(1–13)NH₂ produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1–13)NH₂ nor [Orn⁹]N/OFQ(1–13)NH₂ modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1–13)NH₂ and [Orn⁹]N/OFQ(1–13)NH₂ most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.     

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.     

Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.     

Cystic fibrosis mutations and associated haplotypes in Bulgaria – a comparative population genetic study

We present data on the population genetics of cystic fibrosis (CF) in Bulgaria, obtained by comprehensive mutation analysis and the construction of intragenic microsatellite haplotypes. The sample of 262 CF alleles analysed is representative of the patients diagnosed during the period of referral and of the three main ethnic groups in the country. ΔF508 accounted for 100% of Gypsy CF alleles, which thus differed significantly from both Bulgarians and ethnic Turks. In Bulgarian and Turkish CF patients, 92% of the mutant alleles were identified, yielding a total of 25 different mutations, of which only 7 occurred at frequencies higher than 1%. The findings were compared to other European populations and to the distribution of phenylketonuria mutations. Genetic distances and population trees demonstrated that in the south-eastern tip of Europe, the overall distribution of CF mutations and polymorphic haplotypes is very close to that of Mediterranean populations, with a high frequency of N1303K and G542X, a large number of rare mutations and a prevalence of the 23 31 13 haplotype in association with ΔF508. These findings are consistent with a main role for the Neolithic expansion in the shaping of the CF mutation spectrum in Bulgaria and southern Europe. Received: 1 September 1996     

Humor as a Reward Mechanism: Event-Related Potentials in the Healthy and Diseased Brain

Humor processing involves distinct processing stages including incongruity detection, emotional response, and engagement of mesolimbic reward regions. Dysfunctional reward processing and clinical symptoms in response to humor have been previously described in both hypocretin deficient narcolepsy-cataplexy (NC) and in idiopathic Parkinson disease (PD). For NC patients, humor is the strongest trigger for cataplexy, a transient loss of muscle tone, whereas dopamine-deficient PD-patients show blunted emotional responses to humor. To better understand the role of reward system and the various contributions of hypocretinergic and dopaminergic mechanisms to different stages of humor processing we examined the electrophysiological response to humorous and neutral pictures when given as reward feedback in PD, NC and healthy controls. Humor compared to neutral feedback demonstrated modulation of early ERP amplitudes likely corresponding to visual processing stages, with no group differences. At 270 ms post-feedback, conditions showed topographical and amplitudinal differences for frontal and left posterior electrodes, in that humor feedback was absent in PD patients but increased in NC patients. We suggest that this effect relates to a relatively early affective response, reminiscent of increased amygdala response reported in NC patients. Later ERP differences, corresponding to the late positive potential, revealed a lack of sustained activation in PD, likely due to altered dopamine regulation in reward structures in these patients. This research provides new insights into the temporal dynamics and underlying mechanisms of humor detection and appreciation in health and disease.     

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.