Volume 387, Issue 10_11

Cells are equipped with a number of transcriptional factors that safeguard against various environmental insults. Proteasomal protein degradation plays an important role in the Keap1-Nrf2 cytoprotection system, with molecular machinery similar to that for other environmental defense systems such as inflammatory and hypoxic responses. While Nrf2 protein stabilization is known to be redox-sensitive, the transcription factors NF-κB and HIF-1α for inflammatory and hypoxic responses, respectively, are also influenced by the cellular redox conditions. In this review we present the recently proposed two-site substrate recognition model of the Keap1-Nrf2 system, which regulates the cellular responses against oxidative and xenobiotic stresses. The implications of two destructive motifs in Nrf2, the ETGE and DLG motifs, which appear to function as a hinge and latch attenuating Keap1 activity in different redox states, are discussed.

Sufficient oxygen supply is crucial for the development and physiology of mammalian cells and tissues. When simple diffusion of oxygen becomes inadequate to provide the necessary flow of substrate, evolution has provided cells with tools to detect and respond to hypoxia by upregulating the expression of specific genes, which allows an adaptation to hypoxia-induced stress conditions. The modulation of cell signaling by hypoxia is an emerging area of research that provides insight into the orchestration of cell adaptation to a changing environment. Cell signaling and adaptation processes are often accompanied by rapid and/or chronic remodeling of membrane lipids by activated lipases. This review highlights the bi-directional relation between hypoxia and lipid signaling mechanisms.

Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-κB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.

Reactive oxygen species (ROS) have long been considered only as cyto- and genotoxic. However, there is now compelling evidence that ROS also act as second messengers in response to various stimuli, such as growth factors, hormones and cytokines. The hypoxia-inducible transcription factor (HIF) is a master regulator of oxygen-sensitive gene expression. More recently, HIF has also been shown to respond to non-hypoxic stimuli. Interestingly, recent reports indicate that ROS regulate HIF stability and transcriptional activity in well-oxygenated cells, as well as under hypoxic conditions. Consequently, ROS appear to be key players in regulating HIF-dependent pathways under both normal and pathological circumstances. This review summarizes the current understanding of the role of ROS in the regulation of the mammalian HIF system.

Endothelial dysfunction is an early stage of atherosclerosis and has been attributed to impaired nitric oxide (NO) bioactivity and enhanced formation of oxygen-derived free radicals. Given that endothelial dysfunction is at least in part reversible, the assessment of altered NO availability is of important diagnostic and prognostic significance. Identification of such alterations may help to target asymptomatic individuals who are at risk for cardiovascular diseases and would likely benefit from preventive measures. Focusing on a single signaling pathway, we present here a multi-level approach for the early diagnosis of cardiovascular diseases by assessing molecular, biochemical, structural, and functional changes in the vascular wall.

One of the hallmarks of chronic or severe oxidative stress is the accumulation of oxidized proteins, which tend to form high-molecular-weight aggregates. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasome. This complicated proteolytic system contains a core proteasomal form (20S proteasome) and several regulators. All of these components are affected by oxidative stress to various degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be more resistant. The nuclear proteasome selectively degrades oxidatively damaged histones in the nuclei of mammalian cells, where it is activated and regulated by automodified PARP-1 after oxidative challenge. In this brief review we highlight the proteolysis and its regulatory effects during oxidative stress.

Growing evidence supports the concept that mitochondrial metabolism and reactive oxygen species (ROS) play a major role in aging and determination of an organism's life span. Cellular signaling pathways regulating mitochondrial activity, and hence the generation of ROS and retrograde signaling events originating in mitochondria, have recently moved into the spotlight in aging research. Involvement of the energy-sensing TOR pathway in both mitochondrial signaling and determination of life span has been shown in several studies. This brief review summarizes the recent progress on how mitochondrial signaling might contribute to the aging process with a particular emphasis on TOR signaling from invertebrates to humans.

Hepatic encephalopathy (HE) defines a primary gliopathy associated with acute and chronic liver disease. Astrocyte swelling triggered by ammonia in synergism with different precipitating factors, including hyponatremia, tumor necrosis factor (TNF)-α, glutamate and ligands of the peripheral benzodiazepine receptor (PBR), is an early pathogenetic event in HE. On the other hand, reactive nitrogen and oxygen species (RNOS) including nitric oxide are considered to play a major role in HE. There is growing evidence that osmotic and oxidative stresses are closely interrelated. Astrocyte swelling produces RNOS and vice versa . Based on recent investigations, this review proposes a working model that integrates the pathogenetic action of osmotic and oxidative stresses in HE. Under participation of the N -methyl-D-aspartate (NMDA) receptor, Ca 2+ , the PBR and organic osmolyte depletion, astrocyte swelling and RNOS production may constitute an autoamplificatory signaling loop that integrates at least some of the signals released by HE-precipitating factors.

Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid), DTNB]. For quantification, we also set up an ELISA assay based on the cell-impermeable biotinylated SH reagent, N -(biotinoyl)- N -(iodoacetyl) ethylendiamine (BIAM). Exposure of CHO cells to H 2 O 2 induces oxidation of surface thiols at concentrations not affecting intracellular GSH. Depletion (50%) of GSH decreases surface thiols by 88%. Surface thiols are also highly sensitive to thiol antioxidants, since exposure to 5 mM N - acetyl-L-cysteine (NAC) for 2 h augmented their expression without increasing GSH levels. Using BIAM labeling and two-dimensional gel electrophoresis, we show that this increase in surface thiols is due to the reduction of specific membrane proteins. Peptide mass fingerprinting by MALDI mass spectrometry allowed us to identify two of these proteins as Erp57 and vimentin.

Vascular injury triggers endothelial exocytosis of granules, releasing pro-inflammatory and pro-thrombotic mediators into the blood. Nitric oxide (NO) and reactive oxygen species (ROS) limit vascular inflammation and thrombosis by inhibiting endothelial exocytosis. NO decreases exocytosis by regulating the activity of the N -ethylmaleimide-sensitive factor (NSF), a central component of the exocytic machinery. NO nitrosylates specific cysteine residues of NSF, thereby inhibiting NSF disassembly of the soluble NSF attachment protein receptor (SNARE). NO also modulates exocytosis of other cells; for example, NO regulates platelet activation by inhibiting α-granule secretion from platelets. Other radicals besides NO can regulate exocytosis as well. For example, H 2 O 2 inhibits exocytosis by oxidizing NSF. Using site-directed mutagenesis, we have defined the critical cysteine residues of NSF, and found that one particular cysteine residue, C264, renders NSF sensitive to oxidative stress. Since radicals such as NO and H 2 O 2 inhibit NSF and decrease exocytosis, NSF may act as a redox sensor, modulating exocytosis in response to changes in oxidative stress.

The last decade has witnessed an increased interest in cysteine modifications such as sulfenic and sulfinic acids, thiyl radicals, sulfenyl-amides and thiosulfinates, which come together to enable redox sensing, activation, catalysis, switching and cellular signalling. While glutathionylation, sulfenyl-amide formation and disulfide activation are examples of relatively simple redox responses, the sulfinic acid switch in peroxiredoxin enzymes is part of a complex signalling system that involves sulfenic and sulfinic acids and interacts with kinases and sulfiredoxin. Although the in vivo evaluation of sulfur species is still complicated by a lack of appropriate analytical techniques, research into biological sulfur species has gained considerable momentum and promises further excitement in the future.

Singlet oxygen ( 1 O 2 ), an electronically excited form of molecular oxygen, is a mediator of biological effects of ultraviolet A radiation, stimulating signaling cascades in human cells. We demonstrate here that 1 O 2 generated by photosensitization or by thermodecomposition of 3,3′-(1,4-naphthylidene)dipropionate-1,4-endoperoxide inactivates isolated protein tyrosine phosphatases (PTPases). PTPase activities of PTP1B or CD45 were abolished by low concentrations of 1 O 2 , but were largely restored by post-treatment with dithiothreitol. Electrospray ionization mass spectrometry analysis of tryptic digests of PTP1B exposed to 1 O 2 revealed oxidation of active-site Cys215 as the only cysteine residue oxidized. In summary, 1 O 2 may activate signaling cascades by interfering with phosphotyrosine dephosphorylation.

p66 Shc promotes apoptosis and controls the intracellular redox balance. A fraction of p66 Shc exists within mitochondria, where it oxidizes cytochrome c to form hydrogen peroxide, which in turn induces mitochondrial permeability and apoptosis. However, cells tolerate p66 Shc expression and accumulate oxidative damage under normal conditions, implying that the p66 Shc functions must be tightly regulated. Here we review available knowledge on the regulation of p66 Shc transcription, protein stabilization and post-translational modifications. In addition, we report novel investigations into the role of the mitochondrial import machinery on p66 Shc activation, which highlight the energetic status of mitochondria as a crucial determinant of p66 Shc function.

Air pollution is associated with a variety of respiratory and cardiovascular disorders, including fibrosis. To understand the possible molecular mechanisms underlying this observation, we examined the effect of particulate matter on primary fibroblasts, the key regulators of the extracellular matrix. Fly ash collected in an experimental waste incinerator was used as model particles for fine and ultrafine pollution components. Brief treatment of fibroblasts isolated from adult male Wistar rat hearts with fly ash triggered the immediate formation of intracellular reactive oxygen species (ROS). Using phospho-specific antibodies we observed activation of p38 MAP kinase, p44/42 MAP kinase (ERK1/2) and p70 S6 kinase. Prolonged incubation with fly ash increased the expression of collagen 1 and TGF-β1 , but decreased mRNA levels of MMP9 and TNF-α . Cell proliferation was inhibited at high concentrations of fly ash. An increase in the level of advanced glycation endproduct (AGE) modification of various cellular proteins after long-term treatment of cultured fibroblasts with fly ash was observed. The results of our study demonstrate that direct activation of fibroblasts by combustion-derived particles is a mechanism that may contribute to the adverse health effects of particulate air pollution.

Numerous epidemiological studies have associated exposure to ambient particulate matter (PM) with pulmonary and cardiovascular health effects. Macrophages as a part of the primary pulmonary defence system play a crucial role by generating pro- and anti-inflammatory mediators. The aim of the present study was to examine the effect of incinerator fly ash (MAF02) as a model of environmental particulate matter on the formation of reactive oxygen species (ROS) and their ability to induce oxidative stress in RAW264.7 macrophages. Furthermore, the liberation of arachidonic acid (AA) was observed. The interaction of MAF02 with macrophages caused increased mobilisation of AA, accompanied by enhanced expression of cyclooxygenase-2 (COX-2). The MAF02-induced AA liberation was found to depend on an increased intracellular calcium concentration. In addition, MAF02-induced liberation of AA was selectively blocked by an ERK1/2 pathway-specific inhibitor, while inhibition of the p38 MAPK activity had no effect. Fly ash was also observed to induce an increase in cellular glutathione (GSH) content and antioxidative enzyme haem oxygenase-1 (HO-1). In correlation, experiments with dichlorofluorescein demonstrated increased formation of ROS upon treatment with fly ash. In summary, incinerator fly ash induces oxidative stress to a certain extent, resulting in the onset of important mechanisms related to inflammation.

Proteases are required for the production of peptide neurotransmitters and toxic peptides in neurodegenerative diseases. Unique roles of the cysteine proteases cathepsin L and cathepsin B in secretory vesicles for the production of biologically active peptides have been demonstrated in recent studies. Secretory vesicle cathepsin L participates in the proteolytic conversion of proenkephalin into the active enkephalin, an opioid peptide neurotransmitter that mediates pain relief. Moreover, recent findings provide evidence that cathepsin B in regulated secretory vesicles participates in the production of toxic β-amyloid peptides that are known to accumulate extracellularly in Alzheimer's disease brains. The neurobiological functions of cathepsins L and B demonstrate that these secretory vesicle cysteine proteases produce biologically active peptides. These results demonstrate newly identified roles for cathepsins L and B in neurosecretory vesicles in the production of biologically active peptides.

Two novel metalloendopeptidases in Arabidopsis thaliana , At PreP1 and At PreP2, are responsible for the degradation of targeting peptides in mitochondria and chloroplasts. Both At PreP1 and At PreP2 contain ambiguous targeting peptides and are dually targeted to both organelles. The proteases also have the capacity to degrade unstructured peptides of up to 65 amino acid residues, but not small proteins. The catalysis occurs in a huge catalytic chamber revealed by the crystal structure of At PreP1 at 2.1 Å. The enzymes show a preference for basic and small uncharged amino acids or serines at the cleavage sites. Despite similarities in cleavage specificities, cleavage-site recognition differs for both proteases and is context- and structure-dependent. The At PreP1 and At PreP2 genes are differentially expressed in Arabidopsis .

Taurine is the most abundant free amino acid in heart muscle and protects against heart failure. In the present study, the consequences of hereditary taurine deficiency on cardiac gene expression were examined in 2- and 15–16-month-old taurine transporter knockout ( taut -/- ) mice using a mouse-specific DNA microarray. This oligonucleotide-based microarray contains probes for 251 genes with relevance for heart function. Of these, 163 probes exhibited a reproducible hybridization signal and were analyzed. α-Actin type 1 mRNA levels were 70% lower in the heart of young and older taut -/- mice compared to wild-type controls. Interestingly, the hearts of taut -/- mice showed a switch from α-actin 1 to α-actin 2 expression, as confirmed by real-time PCR and Western blot analysis. In addition, mRNA levels of biomarkers for pressure overload and hypertension were upregulated in taut -/- hearts, i.e., atrial natriuretic factor (+848%), brain natriuretic peptide (+90%), cardiac ankyrin repeat protein (+118%), and procollagen 1a1, 1a2 and 3a1 (+40% at least). These results point to a stress situation in the heart of taut -/- mice under laboratory conditions, and it can be speculated that taut -/- hearts may be even more susceptible to failure in the wild when under exogenous stress.

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.

The molecular architectures of the catalytic core of protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) are similar, and both contain a β12-β13 loop that consists of non-conserved residues. A truncation mutant containing the PP2B catalytic domain has previously been constructed in our laboratory, and designated CNAa. In this study, the PP1 catalytic subunit (PP1c) and CNAa, as well as mutants with the corresponding loops exchanged, were investigated using multiple substrates. Deletion of the β12-β13 loop from Y272 to A279 of PP1c or from Y311 to K318 of CNAa resulted in inactive proteins. Loop exchange generated chimeric mutants called PP1-CNAa-loop and CNAa-PP1-loop. The activities and kinetic parameters of the two chimeric mutants were altered in the direction of the enzyme from which its loop was derived. The activity of PP1c or CNAa-PP1-loop was similar whether preincubated with Mn 2+ or not, while CNAa and PP1-CNAa-loop can acquire enhanced activation if preincubated with Mn 2+ for longer periods of time. Intrinsic fluorescence spectra revealed that the three-dimensional structure was altered as a result of exchanging the loops of PP1c and CNAa. In conclusion, the β12-β13 loop is one of the key regulatory elements in the catalytic domain for the activity and properties of PP1c and CNAa.

The hBRG1 protein, a central ATPase of the human switching/sucrose non-fermenting (SWI/SNF) remodeling complex, has a catalytic ATPase domain, an AT-hook motif and a bromodomain. Bromodomains, found in many chromatin-associated proteins, recognize N -acetyl-lysine in histones and other proteins. The AT-hook motif, first described in the high-mobility group of non-histone chromosomal proteins HMGA1/2, is a DNA-binding motif. The AT-hook binds to the AT-rich DNA sequences in the minor groove of B-DNA in a non-sequence specific manner. AT-hook motifs have been identified in many other DNA-binding proteins. In this study we cloned and purified a fragment of hBRG1 encompassing the AT-hook region and the bromodomain. Nuclear magnetic resonance (NMR) and circular dichroism (CD) analyses show that the recombinant domains are structured. The functionality of subdomains was checked by assessing their interactions with N-acetylated peptides from histones and with DNA. Isothermal titration calorimetric (ITC) analysis demonstrates that the primary micromolar interaction is through the AT-hook motif. The AT-hook region binds to linear DNA by unwinding it. These properties resemble the characteristics of the HMGA1/2 proteins and their interaction with DNA.

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an α/β-hydrolase fold. Here we present the 1.6-Å-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold β-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.

Caenorhabditis elegans is a widely accepted model system for parasitic nematodes, drug screening and developmental studies. Similar to parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins carrying, in part, phosphorylcholine ( P Cho) substitutions, which might play important roles in nematode development, fertility and, at least in the case of parasites, survival within the host. With the exception of a major secretory/excretory product from Acanthocheilonema viteae (ES-62), no protein carrying this epitope has been studied in detail yet. Here we report on the identification, characterization and localization of the aspartyl protease ASP-6 of C. elegans , which is excreted by the nematode in a P Cho-substituted form. Within the worm, most prominent expression of the protein is observed in the intestine, while muscle and epithelial cells express asp-6 to a lesser extent. In animals harboring an ASP-6::GFP fusion protein, diffuse fluorescence throughout the body cavity of adult worms indicates that the chimeric protein is secreted.

The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans . We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans .

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3–S1′ by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1′. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.

The formation of active matrix metalloprotease-2 (MMP-2) requires the proteolytic processing of proMMP-2, a process that can occur through the formation of a ternary complex between proMMP-2, the tissue inhibitor of metalloprotease-2 and membrane type 1-MMP. However, other activation mechanisms have been suggested, and in this study we investigated whether mast cells (MCs) may play a role in the activation of proMMP-2. Murine peritoneal cells, a mixture of macrophages, lymphocytes and MCs, were cultured ex vivo . Addition of proMMP-2 to resting peritoneal cell cultures resulted in only slow conversion of proMMP-2 into the active enzyme. However, when MC degranulation was provoked using a calcium ionophore, proMMP-2 processing was markedly enhanced. When the peritoneal cell populations were depleted in MCs, proMMP-2 processing was abrogated, but was reconstituted when purified MCs were added to the depleted cultures. ProMMP-2 processing was sensitive to serine protease inhibitors, but not to inhibitors of other classes of proteases. Furthermore, proMMP-2 processing was completely abrogated in cells lacking serglycin, a proteoglycan that has previously been shown to mediate storage of a variety of MC serine proteases. Taken together, these results suggest a novel mode of proMMP-2 activation mediated by serglycin-dependent MC serine proteases.