␣-Synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including Parkinson's disease (PD). However, mechanisms that promote intraneuronal ␣-synuclein assembly remain poorly understood. Because pesticides, particularly the herbicide paraquat, have been suggested to play a role as PD risk factors, the hypothesis that interactions between ␣-synuclein and these environmental agents may contribute to aggregate formation was tested in this study. Paraquat markedly accelerated the in vitro rate of ␣-synuclein fibril formation in a dosedependent fashion. When mice were exposed to the herbicide, brain levels of ␣-synuclein were significantly increased. This up-regulation followed a consistent pattern, with higher ␣-synuclein at 2 days after each of three weekly paraquat injections and with protein levels returning to control values by day 7 post-treatment. Paraquat exposure was also accompanied by aggregate formation. Thioflavine S-positive structures accumulated within neurons of the substantia nigra pars compacta, and dual labeling and confocal imaging confirmed that these aggregates contained ␣-synuclein. The results suggest that up-regulation of ␣-synuclein as a consequence of toxicant insult and direct interactions between the protein and environmental agents are potential mechanisms leading to ␣-synuclein pathology in neurodegenerative disorders. Parkinson's disease (PD)1 is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the nigrostriatal pathway and the formation of intraneuronal inclusions (called Lewy bodies) in different brain regions. Although the etiology of PD remains unknown, several lines of evidence suggest a pathogenetic role of the protein ␣-synuclein. In particular, ␣-synuclein is a major component of Lewy bodies in all PD patients (1, 2), and ␣-synuclein mutations have been associated with clinical and pathological parkinsonism in rare autosomal dominant familial cases (3, 4). It has been hypothesized that a tendency of ␣-synuclein to aggregate may underlie its involvement in Lewy body formation and neurodegeneration. In individuals with ␣-synuclein mutations, abnormal forms of the protein could trigger pathological processes as a result of their enhanced propensity to self-assemble (5-7). However, in the vast majority of patients with idiopathic (nonfamilial) PD, the lack of ␣-synuclein mutations (8, 9) indicates that additional mechanisms may lead to conformational changes and consequent protein aggregation. One such mechanism could be the interaction of ␣-synuclein with other chemical species.The association and fibrillation of ␣-synuclein appear to involve a shift in equilibrium from the natively unfolded to a partially folded protein conformation (10). In a recent study, Uversky and colleagues (11) have shown that incubating ␣-synuclein in the presence of paraquat or other pesticides dramatically accelerates the rate of ␣-synuclein fibrillation in vitro, probably due to the preferential binding of these compound...

Parkinson's disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein have been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the alpha-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. To determine whether the presence of GAGs does affect the fibrillation of alpha-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of alpha-synuclein fibrils. Interestingly, the interaction of GAGs with alpha-synuclein is quite specific, since some GAGs, e.g., keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation but also apparently increased the yield of fibrils. The molar ratio of heparin to alpha-synuclein and the incorporation of fluorescein-labeled heparin into the fibrils demonstrate that the heparin is integrated into the fibrils and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating alpha-synuclein fibrillation was 0.19 microM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of alpha-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson's disease.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.Iron-sulfur (Fe-S) clusters are cofactors of proteins involved in oxidation-reduction, electron transport, metabolic conversions, and regulatory functions (1). The iron and sulfur are assembled in fixed stoichiometries (e.g. 2Fe-2S, 4Fe-4S) characteristic of the particular protein and coordinated to critical cysteines in the primary peptide backbone (2). Within cells, iron availability for synthesis of iron-sulfur proteins and other biological functions must be tightly regulated, because excess iron is toxic (3). Excess iron leads to free radical reactions that damage membranes, proteins, and DNA (4). Here we describe a regulatory control mechanism that coordinates iron uptake, iron distribution, and the levels of iron-sulfur cluster proteins in the eukaryote Saccharomyces cerevisiae. The regulator responsible for these effects is Nfs1p.Examination of the S. cerevisiae genome data base reveals that Nfs1p is the single yeast homolog of bacterial IscS (5, 6) and NifS (7). There is strong evidence, both biochemical and genetic, showing that the bacterial protein NifS mobilizes sulfur from cysteine and mediates Fe-S cluster assembly. Bacterial mutants of NifS were found to be deficient in the assembly of both Fe protein and MoFe protein subunits of nitrogenase (8, 9). NifS through its enzymatic activity was found to reactivate the apo form of nitrogenase in which the Fe-S cluster was removed by chelation (10). Elegant biochemical work has elucidated this catalytic process: NifS was shown to be a pyridoxal phosphate-containing homodimer that catalyzes the formation of elemental sulfur from L-cysteine (7). A conserved lysine residue in the bacterial NifS protein (equiva...

BackgroundPhenolic acids are lignin-derived fermentation inhibitors formed during many pretreatment processes of lignocellulosic biomass. In this study, vanillic, p-hydroxybenzoic, and syringic acids were selected as the model compounds of phenolic acids, and the effect of short-term adaptation strategies on the tolerance of S. cerevisiae to phenolic acids was investigated. The mechanism of phenolic acids tolerance in the adapted yeast strains was studied at the morphological and physiological levels.ResultsThe multiple phenolic acids exerted the synergistic inhibitory effect on the yeast cell growth. In particular, a significant interaction between vanillic and hydroxybenzoic acids was found. The optimal short-term adaptation strategies could efficiently improve the growth and fermentation performance of the yeast strain not only in the synthetic media with phenolic acids, but also in the simultaneous saccharification and ethanol fermentation of corncob residue. Morphological analysis showed that phenolic acids caused the parental strain to generate many cytoplasmic membrane invaginations with crack at the top of these sites and some mitochondria gathered around. The adapted strain presented the thicker cell wall and membrane and smaller cell size than those of the parental strain. In particular, the cytoplasmic membrane generated many little protrusions with regular shape. The cytoplasmic membrane integrity was analyzed by testing the relative electrical conductivity, leakage of intracellular substance, and permeation of fluorescent probe. The results indicated that the short-term adaptation improved the membrane integrity of yeast cell.ConclusionThe inhibition mechanism of phenolic acid might be attributed to the combined effect of the cytoplasmic membrane damage and the intracellular acidification. The short-term adaptation strategy with varied stressors levels and adaptive processes accelerated the stress response of yeast cell structure to tolerate phenolic acids. This strategy will contribute to the development of robust microbials for biofuel production from lignocellulosic biomass.

This work expands our tissue proteome capabilities from the analysis of soluble proteins in previous studies to the examination of membrane proteins within the pellets of enriched and selectively isolated tumor cells procured from microdissected tissue specimens. The pellets of targeted ovarian tumor cells are treated by two different membrane protein extraction methods, including the use of detergent and organic solvent. The detergent-based membrane protein preparation protocol not only extracts proteins effectively from cell pellets but also is compatible with subsequent proteome analysis using combined capillary isoelctric focusing/nano reversed-phase liquid chromatography separations coupled with nano electrospray ionization mass spectrometry. Among proteins identified from an amount of pellet equivalent to 20 000 cells, 773 proteins are predicted to contain one or more transmembrane domains, corresponding to 22% membrane proteome coverage within the SwissProt Human protein sequence entries.

We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.

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Cellodextrin transporters (cellodextrin permeases) have been identified in fungi in recent years. However, the functions of these transporters in cellulose utilization and cellulase expression have not been well studied. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum (formally was classified as P. decumbens) were identified, and their functions were analyzed. The deletion of a single cellodextrin transporter gene slightly decreased cellobiose consumption, but no observable effect on cellulase expression was observed, which was attributed to the overlapping activity of isozymes. Further simultaneous deletion of cdtC and cdtD resulted in significantly decreased cellobiose consumption and poor growth on cellulose. The extracellular activity and transcription level of cellulases in the mutant without cdtC and cdtD were significantly lower than those in the wild-type strain when grown on cellulose. This result provides direct evidence of the crucial function of cellodextrin transporters in the induction of cellulase expression by insoluble cellulose.