Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement

Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.     

Influence of nitric oxide on the intracellular reduced glutathione pool: different cellular capacities and strategies to encounter nitric oxide-mediated stress.

Different cell types exhibit huge differences towards the cytotoxic action of NO. In search for an explanation, we used subtoxic concentrations of the NO-donors S-nitrosocysteine (SNOC) for short-term challenge and of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) for longer periods of exposure, respectively, and subtoxic concentrations of the oxidant H2O2 to determine the impact on intracellular reduced glutathione (GSH) concentrations. We find that GSH concentrations are always decreased, but that different cell types show different responses. Incubation of the relatively NO-sensitive murine lymphocytes with both NO-donors, but not with H2O2, resulted in a nearly complete loss of intracellular GSH. Short-term NO-treatment of P815 mastocytoma cells, also sensitive to NO-mediated cell death, decreased GSH to a similar extent only if either glutathione reductase (GSHR) activity or y-glutamylcysteine synthetase (gammaGCS) activity were inhibited concomitantly by specific inhibitors. Long-term NO-treatment of P815 cells, however, resulted in a significant decrease of GSH that could be further enhanced by inhibiting gammaGCS activity. In contrast, neither short-term nor long-term NO-exposure nor H2O2-treatment affected intracellular GSH levels of L929 fibroblasts, which were previously shown to be extremely resistant towards NO, whereas concomitant gammaGCS inhibition, but not GSHR inhibition, completely decreased GSH concentrations. These results show that different cell types use different pathways trying to maintain glutathione concentrations to cope with nitrosative stress, and the overall capability to maintain a critical amount of GSH correlates with susceptibility to NO-induced cell death.     

Mining recent brain proteomic databases for ion channel phosphosite nuggets

Voltage-gated ion channels underlie electrical activity of neurons and are dynamically regulated by diverse cell signaling pathways that alter their phosphorylation state. Recent global mass spectrometric-based analyses of the mouse brain phosphoproteome have yielded a treasure trove of new data as to the extent and nature of phosphorylation of numerous ion channel principal or α subunits in mammalian brain. Here we compile and review data on 347 phosphorylation sites (261 unique) on 42 different voltage-gated ion channel α subunits that were identified in these recent studies. Researchers in the ion channel field can now begin to explore the role of these novel in vivo phosphorylation sites in the dynamic regulation of the localization, activity, and expression of brain ion channels through multisite phosphorylation of their principal subunits.     

Regulation by Ca2+-signaling pathways of adenylyl cyclases

Interplay between the signaling pathways of the intracellular second messengers, cAMP and Ca(2+), has vital consequences for numerous essential physiological processes. Although cAMP can impact on Ca(2+)-homeostasis at many levels, Ca(2+) either directly, or indirectly (via calmodulin [CaM], CaM-binding proteins, protein kinase C [PKC] or Gβγ subunits) may also regulate cAMP synthesis. Here, we have evaluated the evidence for regulation of adenylyl cyclases (ACs) by Ca(2+)-signaling pathways, with an emphasis on verification of this regulation in a physiological context. The effects of compartmentalization and protein signaling complexes on the regulation of AC activity by Ca(2+)-signaling pathways are also addressed. Major gaps are apparent in the interactions that have been assumed, revealing a need to comprehensively clarify the effects of Ca(2+) signaling on individual ACs, so that the important ramifications of this critical interplay between Ca(2+) and cAMP are fully appreciated.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.     

Glutathione and p53 independently mediate responses against oxidative stress in ES cells.

We have investigated the roles of the antioxidant glutathione and p53 in the response of embryonic stem (ES) cells to oxidative stress. ES cells express gammaGCS, a critical enzyme in glutathione (GSH) biosynthesis. Treatment with the pro-oxidant menadione led to elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO, a specific gammaGCS inhibitor depleted GSH pools and prevented the menadione-induced increase in GSH, sensitizing cells to oxidative insult. Although p53 status had no bearing on either the basal levels of GSH or the menadione-induced GSH response, the levels of menadione-induced apoptosis were reduced in the absence of p53. We conclude that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Furthermore, the presence of an intact p53 pathway confers a long-term growth advantage post oxidative stress. Thus, in the absence of p53 ES cells bearing genotoxic damage are less likely to be propagated, suggesting that p53-dependent apoptosis acts to limit the deleterious effects of oxidative stress during early development.     

Survival and cell death in cells constitutively unable to synthesize glutathione

We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.     

Regulation of Endoribonuclease Activity of Alpha-Type Proteasome Subunits in Proerythroleukemia K562 Upon Hemin-Induced Differentiation

The proteasome is the main intracellular proteolytic machine involved in the regulation of numerous cellular processes, including gene expression. In addition to their proteolytic activity, proteasomes also exhibit ATPase/helicase (the 19S particle) and RNAse (the 20S particle) activities, which are regulated by post-translational modifications. In this report we uncovered that several 20S particle subunits: α1 (PSMA6), α2 (PSMA2), α4 (PSMA7), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3) possess RNAse activity against the p53 mRNA in vitro. Furthermore, we found that the RNAse activity of PSMA1 and PSMA3 was regulated upon hemin-induced differentiation of K562 proerythroleukemia cells. The decrease in RNAse activity of PSMA1 and PSMA3 was paralleled by changes in their status of phosphorylation and ubiquitylation. Collectively, our data support the notion that proteasomal RNAse activity may be functionally important and provide insights into the potential mechanism of p53 repression in erythroleukemia cells by RNAse activity of the 20S α-type subunits.     

Anthranilate synthase from Ruta graveolens. Duplicated AS alpha genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.

Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds. The higher plant Ruta graveolens produces tryptophan and elicitor-inducible, anthranilate-derived alkaloids by means of two differentially expressed nuclear genes for chloroplast-localized AS alpha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chorismate between tryptophan and inducible alkaloids thus do not entail chloroplast/cytosol separation of AS isoenzymes and yet might involve differential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutathione S-transferase fusion proteins in Escherichia coli deletion mutants defective in AS activity and were purified to homogeneity. Differential sensitivity of the transformed E. coli strains toward 5-methyltryptophan, a false-feedback inhibitor of AS, was demonstrated. Characterization of affinity-purified AS alpha isoenzymes revealed that the noninducible AS alpha 2 of R. graveolens is strongly feedback inhibited by 10 microns tryptophan. In contrast, the elicitor-inducible AS alpha 1 isoenzyme is only slightly affected even by tryptophan concentrations 10-fold higher than those observed in planta. These results are consistent with the hypothesis that chorismate flux into biosynthesis of tryptophan and defense-related alkaloid biosynthesis in R. graveolens is regulated at the site of AS alpha isoenzymes at both genetic and enzymatic levels.     

蛋白酶体翻译后修饰功能机制研究

蛋白酶体是真核细胞内介导蛋白质特异性降解的主要复合物,在蛋白质质量控制和细胞稳态维持中发挥关键作用.研究发现,蛋白酶体含量或功能的异常会导致癌症、神经退行性疾病等诸多人类恶性疾病.围绕蛋白酶体的活性调控,已经发展了多种靶向药物,加强对蛋白酶体活性精确调控机制的研究具有重要的学术价值与临床意义.蛋白酶体的含量、组装及其活...     

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.     

Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.