Small Heat-stable Protein Research Articles

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.

Differential regulation of glutaredoxin gene expression in response to stress conditions in the yeast Saccharomyces cerevisiae

Glutaredoxins are small heat-stable proteins that are active as glutathione-dependent oxidoreductases and are encoded by two genes, designated GRX1 and GRX2, in the yeast Saccharomyces cerevisiae. We report here that the expression of both genes is induced in response to various stress conditions including oxidative, osmotic, and heat stress and in response to stationary phase growth and growth on non-fermentable carbon sources. Furthermore, both genes are activated by the high-osmolarity glycerol pathway and negatively regulated by the Ras-protein kinase A pathway via stress-responsive STRE elements. GRX1 contains a single STRE element and is induced to significantly higher levels compared to GRX2 following heat and osmotic shock. GRX2 contains two STRE elements, and is rapidly induced in response to reactive oxygen species and upon entry into stationary phase growth. Thus, these data support the idea that the two glutaredoxin isoforms in yeast play distinct roles during normal cellular growth and in response to stress conditions.

Calcium signals in the cell nucleus. Strasbourg, France, August 20-23, 1998.

Just as the great explorations revealed a world far more diverse and rich than had been imagined before, a mass of fast accumulating data is disclosing a much more complex and sophisticated picture of the nucleus than hitherto assumed. The nucleus has long been thought of as some sort of enclosure circumscribed by a double membrane and containing the genetic material of the cell. The inner and outer nuclear membranes join periodically at the nuclear pores, forming complexes supposed to create large channels for the free diffusion of ions and small macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted, with nuclear Ca2+ signals originating from cytosolic Ca2+ waves merely by passive transmission through the nuclear pore complex (NPC). However, recent evidence has changed the mappa mundi of the cell. It has been shown that nuclear Ca2+ can be regulated independently of cytosolic Ca2+ changes. And, just as the Terra Incognita discovered beyond the Ocean Sea proved a fabulous New World, the nuclear envelope (NE) between the cytoplasm and nucleoplasm appears to play an essential role in the regulation of Ca2+ signals inside the nucleus. It contains proteins that regulate and respond to changes in nucleosolic Ca2+ concentration ([Ca2+]nucleosol). Furthermore, several specific Ca2+‐dependent nuclear functions have been described and the list is growing. The regulation of nuclear Ca2+ signals, and the role of nuclear Ca2+ as a specific regulator of nuclear events through Ca2+‐binding proteins, were the main themes of the recent EMBO workshop entitled ‘Calcium signals in the cell nucleus’, organized by A.N.Malviya and coworkers in Strasbourg, France. The existence of Ca2+ signals at the level of the nucleoplasm has never been a real issue, as the …

The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species.

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40-52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using beta-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein-protein interactions.

The catalytic (C) subunit of cAMP-dependent protein kinase interacts with two classes of inhibitors. The regulatory (R) subunits, types I and II, associate to form an inactive holoenzyme complex that is activated in response to cAMP. The C-subunit is also inhibited by small heat-stable protein kinase inhibitors (PKI's). Inhibition by both PKI and RI-subunit requires the synergistic high-affinity binding of MgATP. The stabilizing effect of ATP was quantitated by using analytical gel chromatography. Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent Kd's for subunit association that are below 0.1 nM, while in the absence of MgATP the apparent Kd's are 125 nM and 2.3 microM, respectively, for the two complexes. In the absence of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation. Under physiological salt concentrations, salt-induced dissociation would be substantial in the absence of the high-affinity binding of MgATP. In both complexes, the ATPase activity of the free C-subunit is abolished. The off rates for MgATP also indicate that the type I holoenzyme is more stable than the C.PKI complex. The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h. When the C.PKI complex is incubated with RI-subunit in the presence or absence of MgATP, the C-subunit preferentially reassociates with the RI-subunit, forming holoenzyme. In contrast, free PKI cannot compete for the C-subunit when it is part of a holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the Gin recombination protein of Escherichia coli phage Mu and its host factor

Inversion of the G-segment of Escherichia coli phage Mu was studied in vitro. The reaction requires the Gin recombination protein, which was purified to near homogeneity from overproducing cells. Upon purification the protein lost activity, which was restored by addition of an extract from uninfected E. coli cells. The stimulatory host factor is a small heat-stable protein and was purified from E. coli cells. Full recombination required both proteins, but Gin alone promoted some recombination by itself, particularly at high concentrations. Relaxation of negative supercoils and recombination of a substrate with two recombination sites in an inverted orientation both have the same specificity for Gin and the host factor. The Gin-associated topoisomerase activity appears tightly coupled to its recombination activity.

44] Purification and properties of the ATPase inhibitor from bovine heart mitochondria

Publisher Summary This chapter discusses the methods for the purification and highlights the properties of the ATPase Inhibitor from bovine heart mitochondria. These methods are an improvement over the original procedure with respect to yield and purity. Some new aspects of the properties of the inhibitor are also described. Mitochondrial ATPase (F1) inhibitor is a small heat-stable protein. When complexed with either soluble or membrane-bound F1, the protein inhibits the ATPase activity of the enzyme, but does not interfere with F1-dependent oxidative phosphorylation. The inhibitor-F1 complex that exhibits no ATPase activity retains an undiminished capacity to restore oxidative phosphorylation in F1-deficient submitochondrial particles (SMP). The inhibitor content of fractions emerging from the purification procedure is determined by measuring the inhibition of the ATPase activity of either submitochondrial particles or soluble F1.

Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.

Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system in Salmonella typhimurium.

Adenylate cyclase (EC 4.6.1.1) and several carbohydrate permeases are inhibited by D-glucose and other substrates of the phosphoenolpyruvate:sugar phosphotransferase system. These activities are coordinately altered by sugar substrates of the phosphotransferase system in a variety of bacterial strains which contain differing cellular levels of the protein components of the phosphotransferase system: Enzyme I, a small heat-stable protein, and Enzyme II. It is suggested that the activities of adenylate cyclase and the permease proteins are subject to allosteric regulation and that the allosteric effector is a regulatory protein which can be phosphorylated by the phosphotransferase system.

Physiological Basis of Transient Repression of Catabolic Enzymes in Escherichia coli

Transient repression of catabolic enzymes occurs in cells that encounter a new carbon compound in their growth medium, but only when the cells contain the enzyme catalyzing the transfer of phosphate from phosphoenolpyruvate to a small heat-stable protein (HPr), as well as a permease capable of transporting the new compound across the cell membrane. The newly added compound need not be metabolized. The degree and duration of the transient repression have no obvious relation to the intracellular level of the exogenously added compound. It is suggested that the actual passage of the compound through the cell membrane is responsible for the repression.

Two classes of pleiotropic mutants of Aerobacter aerogenes lacking components of a phosphoenolpyruvate-dependent phosphotransferase system.

where PEP denotes phosphoenolpyruvate; HPr, a small heat-stable protein containing histidine; and P-HPr, the phosphorylated protein. Their data indicated that I and HPr have broad functions serving a family of enzymes II, each specific for one or a few 2 We have recently demonstrated that the dissimilation of D-mannitol by Aerobacter aerogenes depends upon a phosphotransferase system similar to the one described above. In the absence of an inducible specific for D-mannitol (referred to as enzyme II in this work), A. aerogenes fails to grow on this compound.3 In the present report some of the phenotypic consequences of mutations affecting I or HPr are described. In contradistinction to the loss of II, which renders the cell incapable of utilizing a particular compound, the lack of I or HPr pleiotropically affects the metabolism of a number of polyhydric alcohols and sugars. Materials and Methods.-Bacteria: Strain 5P14, a double auxotroph of A. aerogenes 1033 (ref. 4), provided by B. Magasanik, was used as the starting cell line. Its strict requirement for arginine and guanine permitted distinction of mutant progeny from contaminating organisms. For our convenience this strain has been called strain 2002 (ref. 3) and will be referred to as such in this communication. Strain 2006, a mutant lacking II, hence unable to grow on.Dmannitol, was obtained from strain 2002 (ref. 3). Strain 2050, lacking I, and strain 2070, lacking HPr, were both isolated as D-mannitol-negative clones from populations of strain 2002 treated with ethyl methanesulfonate.5 MacConikey agar in which D-mannitol was substituted for lactose6 was used for the detection of these mutants. Strain 2051, a spontaneous revertant exhibiting normal growth rate on agar with D-mannose as carbon source, was derived from a population of cells of strain 2050 recycled in liquid medium with the same carbon source. Strain 2071 was isolated as a spontaneous revertant by directly plating 109 cells of strain 2070 on agar with D-sorbitol as carbon source. Chemicals: _-C14-D-mannitol (25 c/mole) was obtained from Nuclear-Chicago Corp.; Dmannitol, L-arabinose, D-mannose, and D-fructose from Eastman Kodak Co.; glycerol and maltose from Fisher Scientific Co.; D)-arabitol, ribitol, D-sorbitol, D-ribose, and D-galactose from Pfanstiehl Chemical Co.; i-inositol, lactose, and acid-hydrolyzed casein from Nutritional Biochemicals Corp.; D-glucose from Merck and Co.; and PEP (sodium salt) from Calbiochem.