Regulation of protein S-thiolation by glutaredoxin 5 in the yeast Saccharomyces cerevisiae

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein -SH groups form mixed disulfides with low molecular weight thiols such as glutathione. We report here that this protein modification is not a simple response to the cellular redox state, since different oxidants lead to different patterns of protein S-thiolation. SDS-polyacrylamide gel electrophoresis shows that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the major target for modification following treatment with hydroperoxides (hydrogen peroxide or tert-butylhydroperoxide), whereas this enzyme is unaffected following cellular exposure to the thiol oxidant diamide. Further evidence that protein S-thiolation is tightly regulated in response to oxidative stress is provided by the finding that the Tdh3 GAPDH isoenzyme, and not the Tdh2 isoenzyme, is S-thiolated following exposure to H(2)O(2) in vivo, whereas both GAPDH isoenzymes are S-thiolated when H(2)O(2) is added to cell-free extracts. This indicates that cellular factors are likely to be responsible for the difference in GAPDH S-thiolation observed in vivo rather than intrinsic structural differences between the GAPDH isoenzymes. To begin to search for factors that can regulate the S-thiolation process, we investigated the role of the glutaredoxin family of oxidoreductases. We provide the first evidence that protein dethiolation in vivo is regulated by a monothiol-glutaredoxin rather than the classical glutaredoxins, which contain two active site cysteine residues. In particular, glutaredoxin 5 is required for efficient dethiolation of the Tdh3 GAPDH isoenzyme.     

Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability.     

Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Simulated actin reorganization mediated by motor proteins

Cortical actin networks are highly dynamic and play critical roles in shaping the mechanical properties of cells. The actin cytoskeleton undergoes significant reorganization in many different contexts, including during directed cell migration and over the course of the cell cycle, when cortical actin can transition between different configurations such as open patched meshworks, homogeneous distributions, and aligned bundles. Several types of myosin motor proteins, characterized by different kinetic parameters, have been involved in this reorganization of actin filaments. Given the limitations in studying the interactions of actin with myosin in vivo, we propose stochastic agent-based models and develop a set of data analysis measures to assess how myosin motor proteins mediate various actin organizations. In particular, we identify individual motor parameters, such as motor binding rate and step size, that generate actin networks with different levels of contractility and different patterns of myosin motor localization, which have previously been observed experimentally. In simulations where two motor populations with distinct kinetic parameters interact with the same actin network, we find that motors may act in a complementary way, by tuning the actin network organization, or in an antagonistic way, where one motor emerges as dominant. This modeling and data analysis framework also uncovers parameter regimes where spatial segregation between motor populations is achieved. By allowing for changes in kinetic rates during the actin-myosin dynamic simulations, our work suggests that certain actin-myosin organizations may require additional regulation beyond mediation by motor proteins in order to reconfigure the cytoskeleton network on experimentally-observed timescales.     

Limnological Characteristics of Two Eutrophic and Four Mesotrophic Lakes in West-central Florida

Limnological characteristics of six subtropical lakes were monitored to determine the factors which regulate chlorophyll α concentrations and phytoplankton standing crops. Most physical chemical variables showed non-significant differences with depth but differences between lakes often were large. Phytoplankton blooms occurred throughout the year and there were marked differences between the hypereutrophic and mesotrophic lakes in chlorophyll α concentrations, standing stocks, and dominant species. Densities of total zooplankton and rotifers in the hypereutrophic lakes were 3- to 6-fold greater than in the mesotrophic lakes. Regression models for chlorophyll α and total phytoplankton cell volume were calculated for each lake and for all lakes combined, but R² values and numbers of shared variables tended to be low indicating the need for additional variables and more frequent sampling.     

Toxicity of Linoleic Acid Hydroperoxide to Saccharomyces cerevisiae: Involvement of a Respiration-Related Process for Maximal Sensitivity and Adaptive Response

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho⁰] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho⁰] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 μg ml⁻¹). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37°C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.