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A tryptophan mutant of Sup35 ([NM.sup.F117W]) is used to probe aggregation of the Sup35 protein of Saccharomyces cerevisiae which forms a prion that generates the [PSI(+)] phenotype and also gain insight into the NM region that governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. The results provide insight into the temperature-insensitive nucleation and temperature dependent fibril extension and the effects of temperature on the aggregation of the Sup35 NM domain could be dissected by introduction of Trp 117.

Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent Km values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with Vmax values of 0.294 and 0.173 μmol mg−1 min−1, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the Km for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the Vmax changed from 0.806 to 0.673 μmol mg−1 min−1. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate. [ABSTRACT FROM AUTHOR]

We studied the effect of the loss of the Ser- Thr protein phosphatase Sit4, an important post-translational regulator, on the steady-state levels of the low-affinity glucose transporter Hxt1p and observed a delay in its appearance after high glucose induction, slow growth, and diminished glucose consumption. By analyzing the known essential pathway necessary to induce Hxt1p, we observed a partial inhibition of casein kinase I activity. In both WT and sit4Δ strains, the transcript was induced with no significant difference at 15 min of glucose induction; however, after 45 min, a clear difference in the level of expression was observed being 45% higher in WT than in sit4Δ strain. As at early time of induction, the HXT1 transcript was present but not the protein in the sit4Δ strain we analyzed association of HXT1 with ribosomes, which revealed a significant difference in the association profile; in the mutant strain, the HXT1 transcript associated with a larger set of ribosomal fractions than it did in the WT strain, suggesting also a partial defect in protein synthesis. Overexpression of the translation initiation factor TIF2/ eIF4A led to an increase in Hxt1p abundance in the WT strain only. It was concluded that Sit4p ensures that HXT1 transcript is efficiently transcribed and translated thus increasing protein levels of Hxt1p when high glucose levels are present. [ABSTRACT FROM AUTHOR]

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROSsensitive fluorescent probes. Heme-induced ROS production was time-and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism. [ABSTRACT FROM AUTHOR]

A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. PathologicaI signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, lithium induces a ‘galactosemia-like’ phenotype as a consequence of inhibition of phosphoglucomutase, a key enzyme in galactose metabolism. Induced galactose toxicity is prevented by deletion of GAL4, which inhibits the transcriptional activation of genes involved in galactose metabolism and by deletion of the galactokinase ( GAL1), indicating that galactose-1-phosphate, a phosphorylated intermediate of the Leloir pathway, is the toxic compound. As an alternative to inhibiting entry and metabolism of galactose, we investigated whether deviation of galactose metabolism from the Leloir pathway would also overcome the galactosemic effect of lithium. We show that cells overexpressing the aldose reductase GRE3, which converts galactose to galactitol, are more tolerant to lithium than wild-type cells when grown in galactose medium and they accumulate more galactitol and less galactose-1-phosphate. Overexpression of GRE3 also suppressed the galactose growth defect of the ‘galactosemic’ gal7- and gal10-deleted strains, which lack galactose-1-P-uridyltransferase or UDP-galactose-4-epimerase activities, respectively. Furthermore, the effect of GRE3 was independent of the inositol monophosphatases INM1 and INM2. We propose that lithium induces a galactosemic state in yeast and that inhibition of the Leloir pathway before the phosphorylation step or stimulation of galactitol production suppresses lithium-induced galactose toxicity. [ABSTRACT FROM AUTHOR]