Recent work from several laboratories has demonstrated that proteolytic mechanisms signi®cantly contribute to the molecular interplay between Streptococcus pyogenes, an important human pathogen, and its host. Here we describe the identi®cation, puri®cation and characterization of a novel extracellular cysteine proteinase produced by S.pyogenes. This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme of S.pyogenes, is distinct from the well-characterized streptococcal cysteine proteinase, SpeB, and cleaves human IgG in the hinge region with a high degree of speci®city. Thus, other human proteins, including immunoglobulins M, A, D and E, are not degraded by IdeS. The enzyme ef®ciently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing of S.pyogenes by phagocytic cells. This and additional observations on the distribution and expression of the ideS gene indicate that IdeS represents a novel and signi®cant bacterial virulence determinant, and a potential therapeutic target.
In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure. Molecular & Cellular Proteomics 7:1489 -1500, 2008.A key element of the experimental framework for systems biology is the comprehensive, quantitative measurement of whole biological systems in differentially perturbed states (1). Among the different types of measurements possible, protein quantification is particularly informative because proteins catalyze or control the majority of cellular functions. Currently the most widely applied quantitative proteome analysis technologies consist of the labeling of the samples by stable isotopes, the reproducible separation of complex peptide mixtures, usually by capillary LC, and the identification and quantification of selected peptides by tandem mass spectrometry and sequence database searching (2, 3). Relative quantitative values are generated by these methods if two or more samples are being compared, and absolute quantification can be achieved if suitable, calibrated reference samples are available (4). Using such shotgun methods, in each measurement only a fraction of the analytes present in a complex sample is identified and quantified. Peptide ions are selected by the mass spectrometer automatically based on precursor ion signal intensities. Due to a multitude of factors, including interference between analytes and variations in precursor ion spectra, the selection of peptides is not reproducible in consecutive runs in particular for peptides...
SummaryDuring the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1 Δ Δ Δ Δ cyc7 Δ Δ Δ Δ and cyc3 Δ Δ Δ Δ , both lacking mature cytochrome c , displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c .
In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD ؉ . In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g ؊1 ) and the lowest xylitol yield (0.05 g g ؊1 ) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.Fuel ethanol produced from fermentation of lignocellulosic hydrolysates is an attractive replacement for liquid fossil fuels because its production is renewable and it does not generate net carbon dioxide. Hydrolysis of lignocellulose generates mostly hexose but also some pentose sugars. The pentose sugars cannot be metabolized by Saccharomyces cerevisiae, the preferred ethanol-producing microorganism. In hydrolysate made from hardwood, xylose must be fermented to ethanol for the process to be economically feasible (39). The yeast Pichia stipitis metabolizes xylose through expression of the XYL1 gene, encoding xylose reductase (XR), and the XYL2 gene, encoding xylitol dehydrogenase (XDH). XR catalyzes the reduction of xylose to xylitol by using NADH or NADPH (30), whereas XDH oxidizes xylitol to xylulose exclusively with NAD ϩ (31). Unfortunately, P. stipitis is sensitive to ethanol (10) and requires low and carefully controlled oxygenation (34), which prevents its use for industrial ethanol production.Recombinant S. cerevisiae strains expressing the XYL1 and XYL2 genes from P. stipitis have been constructed and can ferment xylose (26); however, most of the consumed xylose is secreted as xylitol (20,26,36,42). Xylitol production can be lowered by overexpression of the XKS1 gene, which encodes the native xylulokinase (XK) (23), but still about one third of the consumed xylose is converted to xylitol under anaerobic conditions (13). Xylitol formation may result from the cofactor imbalance between the ...
Mitochondrial involvement in yeast apoptosis is probably the most unifying feature in the field. Reports proposing a role for mitochondria in yeast apoptosis present evidence ranging from the simple observation of ROS accumulation in the cell to the identification of mitochondrial proteins mediating cell death. Although yeast is unarguably a simple model it reveals an elaborate regulation of the death process involving distinct proteins and most likely different pathways, depending on the insult, growth conditions and cell metabolism. This complexity may be due to the interplay between the death pathways and the major signalling routes in the cell, contributing to a whole integrated response. The elucidation of these pathways in yeast has been a valuable help in understanding the intricate mechanisms of cell death in higher eukaryotes, and of severe human diseases associated with mitochondria-dependent apoptosis. In addition, the absence of obvious orthologues of mammalian apoptotic regulators, namely of the Bcl-2 family, favours the use of yeast to assess the function of such proteins. In conclusion, yeast with its distinctive ability to survive without respiration-competent mitochondria is a powerful model to study the involvement of mitochondria and mitochondria interacting proteins in cell death.
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