Pathway Correlation Profile of Gene-Gene Co-Expression for Identifying Pathway Perturbation

Identifying perturbed or dysregulated pathways is critical to understanding the biological processes that change within an experiment. Previous methods identified important pathways that are significantly enriched among differentially expressed genes; however, these methods cannot account for small, coordinated changes in gene expression that amass across a whole pathway. In order to overcome this limitation, we use microarray gene expression data to identify pathway perturbation based on pathway correlation profiles. By identifying the distribution of gene-gene pair correlations within a pathway, we can rank the pathways based on the level of perturbation and dysregulation. We have shown this successfully for differences between two experimental conditions in Escherichia coli and changes within time series data in Saccharomyces cerevisiae, as well as two estrogen receptor response classes of breast cancer. Overall, our method made significant predictions as to the pathway perturbations that are involved in the experimental conditions.     

Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae

Glyoxylate biosynthesis in Saccharomyces cerevisiae is traditionally mainly ascribed to the reaction catalyzed by isocitrate lyase (Icl), which converts isocitrate to glyoxylate and succinate. However, Icl is generally reported to be repressed by glucose and yet glyoxylate is detected at high levels in S. cerevisiae extracts during cultivation on glucose. In bacteria there is an alternative pathway for glyoxylate biosynthesis that involves a direct oxidation of glycine. Therefore, we investigated the glycine metabolism in S. cerevisiae coupling metabolomics data and (13)C-isotope-labeling analysis of two reference strains and a mutant with a deletion in a gene encoding an alanine:glyoxylate aminotransferase. The strains were cultivated on minimal medium containing glucose or galactose, and (13)C-glycine as sole nitrogen source. Glyoxylate presented (13)C-labeling in all cultivation conditions. Furthermore, glyoxylate seemed to be converted to 2-oxovalerate, an unusual metabolite in S. cerevisiae. 2-Oxovalerate can possibly be converted to 2-oxoisovalerate, a key precursor in the biosynthesis of branched-chain amino acids. Hence, we propose a new pathway for glycine catabolism and glyoxylate biosynthesis in S. cerevisiae that seems not to be repressed by glucose and is active under both aerobic and anaerobic conditions. This work demonstrates the great potential of coupling metabolomics data and isotope-labeling analysis for pathway reconstructions.     

酿酒酵母促分裂原蛋白激酶Hog1p 介导的渗透胁迫反应调控机制

 高渗透性甘油促分裂原激酶信号转导途径（high osmolarity glycerol mitogen activated protein kinase signaling transduction pathway,HOG-MAPK）是调控酿酒酵母对外界高渗透压胁迫环境应答的主要途径,促分裂原蛋白激酶Hog1p（MAPK Hog1p）是其中的关键性作用因子.在高渗透压刺激时,MAPK Hog1p接受信号被特异性激活并进入核内,调控相关胁迫应答基因的表达,并介导该时期细胞周期的阻滞,从而增强细胞对外界不利环境的适应能力.对胁迫条件下酿酒酵母中MAPK Hog1p作用机制的进一步研究,有利于更深入地了解哺乳动物体内逆境激发促分裂原蛋白激酶途径的功能和调控机制.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

Modeling and stochastic simulation of the Ras/cAMP/PKA pathway in the yeast Saccharomyces cerevisiae evidences a key regulatory function for intracellular guanine nucleotides pools

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of metabolism and cell cycle progression. The pathway is tightly regulated by several control mechanisms, as the feedback cycle ruled by the activity of phosphodiesterase. Here, we present a discrete mathematical model for the Ras/cAMP/PKA pathway that considers its principal cytoplasmic components and their mutual interactions. The tau-leaping algorithm is then used to perform stochastic simulations of the model. We investigate this system under various conditions, and we test how different values of several stochastic reaction constants affect the pathway behaviour. Finally, we show that the level of guanine nucleotides, GTP and GDP, could be relevant metabolic signals for the regulation of the whole pathway.     

Integration of group II intron bI1 into a foreign RNA by reversal of the self-splicing reaction in vitro

Group II intron bI1, the first intron of the COB gene in the mitochondria of S. cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing. We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction. The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew. When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence. This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.     

Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid

Saccharomyces cerevisiae is known to be able to adapt to the presence of the commonly used food preservative benzoic acid with a large energy expenditure. Some mechanisms for the adaptation process have been suggested, but its quantitative energetic and metabolic aspects have rarely been discussed. This study discusses use of the stimulus response approach to quantitatively study the energetic and metabolic aspects of the transient adaptation of S. cerevisiae to a shift in benzoic acid concentration, from 0 to 0.8 mM. The information obtained also serves as the basis for further utilization of benzoic acid as a tool for targeted perturbation of the energy system, which is important in studying the kinetics and regulation of central carbon metabolism in S. cerevisiae. Using this experimental set-up, we found significant fast-transient (< 3000 s) increases in O(2) consumption and CO(2) production rates, of approximately 50%, which reflect a high energy requirement for the adaptation process. We also found that with a longer exposure time to benzoic acid, S. cerevisiae decreases the cell membrane permeability for this weak acid by a factor of 10 and decreases the cell size to approximately 80% of the initial value. The intracellular metabolite profile in the new steady-state indicates increases in the glycolytic and tricarboxylic acid cycle fluxes, which are in agreement with the observed increases in specific glucose and O(2) uptake rates.     

Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation

The cAMP-protein kinase A (PKA) pathway is a major signalling pathway in the yeast Saccharomyces cerevisiae, but also in many other eukaryotic cell types, including mammalian cells. Since cAMP plays a crucial role as second messenger in the regulation of this pathway, its levels are strictly controlled, both in the basal condition and after induction by agonists. A major factor in the down-regulation of the cAMP level after stimulation is PKA itself. Activation of PKA triggers feedback down-regulation of the increased cAMP level, stimulating its return to the basal concentration. This is accomplished at different levels. The best documented mechanisms are: inhibition of cAMP synthesis by down-regulation of adenylate cyclase and/or its regulatory proteins, stimulation of cAMP breakdown by phosphodiesterases and spatial regulation of cAMP levels in the cell by A-Kinase Anchoring Proteins (AKAPs). In this review we describe these processes in detail for S. cerevisiae, for cells of mammals and selected other organisms, and we hint at other possible targets for feedback regulation of intracellular cAMP levels.     

Mitochondria inheritance in yeast saccharomyces cerevisiae

The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.     

New aspects on phosphate sensing and signalling in Saccharomyces cerevisiae

The mechanism involved in the cellular phosphate response of Saccharomyces cerevisiae forms part of the PHO pathway, which upon expression allows a co-ordinated cellular response and adaptation to changes in availability of external phosphate. Although genetic studies and analyses of the S. cerevisiae genome have produced much information on the components of the PHO pathway, little is known about how cells sense the environmental phosphate level and the mechanistic regulation of phosphate acquisition. Recent studies emphasize different levels in phosphate sensing and signalling in response to external phosphate fluctuations. This review integrates all these findings into a model involving rapid and long-term effects of phosphate sensing and signalling in S. cerevisiae. The model describes in particular how yeast cells are able to adjust phosphate acquisition by integrating the status of the intracellular phosphate pools together with the extracellular phosphate concentration.     

Protein targeting to and translocation across the membrane of the endoplasmic reticulum.

Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum. Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae. Results obtained in each of these systems have contributed novel, mostly non-overlapping information. Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon. Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S. cerevisiae, by selecting for mutants in the translocation pathway. None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems. In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.     

A mathematical model of the role of aggregation in sonic hedgehog signalling

Effective regulation of the sonic hedgehog (Shh) signalling pathway is essential for normal development in a wide variety of species. Correct Shh signalling requires the formation of Shh aggregates on the surface of producing cells. Shh aggregates subsequently diffuse away and are recognised in receiving cells located elsewhere in the developing embryo. Various mechanisms have been postulated regarding how these aggregates form and what their precise role is in the overall signalling process. To understand the role of these mechanisms in the overall signalling process, we formulate and analyse a mathematical model of Shh aggregation using nonlinear ordinary differential equations. We consider Shh aggregate formation to comprise of multimerisation, association with heparan sulfate proteoglycans (HSPG) and binding with lipoproteins. We show that the size distribution of the Shh aggregates formed on the producing cell surface resembles an exponential distribution, a result in agreement with experimental data. A detailed sensitivity analysis of our model reveals that this exponential distribution is robust to parameter changes, and subsequently, also to variations in the processes by which Shh is recruited by HSPGs and lipoproteins. The work demonstrates the time taken for different sized Shh aggregates to form and the important role this likely plays in Shh diffusion.     

Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae)

Toxicities of the radiolytically generated oxidizing radicals HO(*), CO(3)(-)(*), and NO(2)(*) toward suspension cultures of a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae) were examined. As demonstrated by the absence of protection from the membrane-impermeable HO(*) scavenger polyethylene glycol (PEG), externally generated HO(*) was not bactericidal under these conditions; however, partial protection by PEG was observed for S. cerevisiae, indicating the presence of a fungicidal pathway involving external HO(*). For both organisms, conversion of external HO(*) to the secondary radical, CO(3)(-)(*), by reaction with HCO(3)(-) increases their susceptibility to radiolytic killing. In contrast, externally generated NO(2)(*) exhibited toxicity comparable to that of CO(3)(-)(*) toward E. coli, but completely blocked the extracellular toxicity of HO(*) toward S. cerevisiae. Cogeneration of equal fluxes of NO(2)(-)(*) and either HO(*) or CO(3)(-)(*) also essentially eliminated the extracellular microbicidal reactions. This behavior is consistent with expectations based upon relative rates of radical-radical self-coupling and cross-coupling reactions. The different patterns of toxicity observed imply fundamentally different microbicidal mechanisms for the two organisms, wherein the bacterium is susceptible to killing by oxidation of highly reactive targets on its cellular envelope but, despite undergoing similar oxidative insult, the fungus is not.