Labile Protein Research Articles

Regulation of surface coat exchange by differentiating African trypanosomes

African trypanosomes ( Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 μM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca 2+ ([Ca 2+] i). LMS in mercury-treated cells that had been pre-incubated with a specific Ca 2+-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca 2+-signaling pathway. Exposure to 20 μM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca 2+] i, suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations < 400 μM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca 2+] I, therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.

SOCS1 Inhibits Tumor Necrosis Factor-induced Activation of ASK1-JNK Inflammatory Signaling by Mediating ASK1 Degradation

We have previously shown that ASK1 undergoes ubiquitination and degradation in resting endothelial cells (EC) and that proinflammatory cytokine tumor necrosis factor (TNF) induces deubiquitination and stabilization, leading to ASK1 activation. However, the mechanism for the regulation of ASK1 stability is not known. In the present study, we have shown that SOCS1, a member of suppressor of cytokine signaling, induces ASK1 degradation. SOCS1 was constitutively expressed in EC and formed a labile complex with ASK1 that can be stabilized by proteasomal inhibitors. The phosphotyrosine-binding SH2 domain of SOCS1 was critical for its association with ASK1. Thus a SOCS1 mutant defective in phosphotyrosine binding failed to bind to and induce ASK1 degradation. Phosphotyrosine of ASK1 was induced in response to growth factors, and TNF induced dephosphorylation and dissociation of ASK1 from SOCS1. ASK1 with a mutation at Tyr-718 diminished the binding to SOCS1, suggesting that the phosphotyrosine-718 of ASK1 is critical for SOCS1 binding. Moreover, ASK1 expression and activity were up-regulated in SOCS1-deficient mice and derived EC, resulting in enhanced TNF-induced activation of JNK, expression of proinflammatory molecules, and apoptotic responses. We concluded that SOCS1 functions as a negative regulator in TNF-induced inflammation in EC, in part, by inducing ASK1 degradation.

Lipopolysaccharide Induces Epithelium- and Prostaglandin E2-Dependent Relaxation of Mouse Isolated Trachea through Activation of Cyclooxygenase (COX)-1 and COX-2

Lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 agonist, causes airway hyperreactivity through nuclear factor-kappaB (NF-kappaB). Because NF-kappaB induces cyclooxygenase-2 (COX-2) to increase synthesis of prostaglandins (PGs), including the potent airway anti-inflammatory and smooth muscle relaxant PGE(2), we investigated whether LPS causes short-term PGE(2)-dependent relaxation of mouse isolated trachea. In rings of trachea contracted submaximally with carbachol, LPS caused slowly developing, epithelium-dependent relaxations that reached a maximum within 60 min. Fluorescence immunohistochemistry revealed TLR4-like immunoreactivity localized predominantly to the epithelium. The LPS antagonist polymixin B; the nonselective COX inhibitor indomethacin; the selective COX-1 and COX-2 inhibitors 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560) and 4-[5-(4-chlorophenyl)-1-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfonamide (SC236), respectively; the transcription inhibitor actinomycin D; the translation inhibitor cycloheximide; the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imadazole (SB203580); and a combination of the mixed DP/EP1/EP2 receptor antagonist 6-isopropoxy-9-xanthone-2-carboxylic acid (AH6809) and the EP4 receptor antagonist 4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1-5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide (L-161982) all abolished relaxation to LPS, giving instead slowly developing, small contractions over 60 min. The cytosolic phospholipase A(2) (cPLA(2)) inhibitor 1,1,1-trifluoro-6Z,9Z, 12Z,15Z-heneicosateraen-2-one significantly (p < 0.05) inhibited the relaxation to LPS, whereas the NF-kappaB proteasomal inhibitor Z-Leu-Leu-Leu-aldehyde (MG-132) had no affect on the relaxation in the first 20 min, after which it reversed the response to a contraction. In conclusion, our data indicate that LPS activates airway epithelial TLR4 to cause release of PGE(2) and subsequent EP2 and EP4 receptor-dependent smooth muscle relaxation. Activation of both COX-1 and COX-2 seems to be essential for this novel response to LPS, which also involves cPLA(2), p38 MAPK, NF-kappaB, and an unidentified NF-kappaB-independent, labile regulatory protein.

High-solids centrifuge is a boon and a curse for managing anaerobically digested biosolids

High-solids centrifugation can reduce the cost of managing or disposing of anaerobically digested biosolids. High-solids centrifuges can increase relative cake solids by as much as 5% DS compared with other dewatering devices, such as belt filter presses, with a resulting 15-20% reduction in overall mass of hauled biosolids. Cost reductions can be similar (15-20%) or more, depending on the type of disposal or management involved. For example, the additional removal of water from the cake increases the energy content in the biosolids, thereby facilitating incineration or heat drying processes. For land application, the benefits are more mixed. As explained in this paper, increases in biosolids odours associated with high-solids centrifuges may increase digestion requirements and may compel producers to transport biosolids to more remote, distant sites, potentially increasing transportation costs. High-solids centrifuges shear anaerobically digested biosolids. The shear results in a net increase in labile protein, an odour precursor. Additionally, high-solids centrifugation also results in the inhibition of methanogenesis, a major mechanism for degradation of organosulphur odours. Therefore, the risks and benefits should both be weighed when considering high-solids centrifuges for land application of anaerobically digested biosolids.

On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum

BackgroundCycloheximide is a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has previously been shown that a short incubation of Dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis.ResultsWe found that treatment with cycloheximide also causes the amoebae to retract their pseudopodia, round up and cease movement. Furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of 2 mM cycloheximide, although membrane uptake, as measured using FM1-43, is unaffected. In the presence of cycloheximide, aggregation-competent amoebae sensitive to cAMP, although round, can still localise CRAC, ABP120, PI3K and actin polymerisation in response to a micropipette filled with cAMP. The behaviour of wild-type amoebae in the presence of cycloheximide is surprisingly similar to that of amoebae having a temperature-sensitive version of NSF at the restrictive temperature.ConclusionOur results may suggest that, upon cycloheximide treatment, either a labile protein required for polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated.

The Carboxy-Terminal Neh3 Domain of Nrf2 Is Required for Transcriptional Activation

Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal "Neh3" domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

Anthropogenic influence on the organic fraction of sediments in two contrasting estuaries: A biochemical approach

Abundance and biochemical composition of organic materials in sediments of the estuaries of Mundaka and Bilbao (SE Bay of Biscay) were analysed to assess the effect of organic wastes released to these systems. Organic and labile contents were higher in the sediments of Bilbao, denoting organic enrichment with poorly decomposed materials by human dumping. Spatially, organic matter distribution skewed seaward in Bilbao and upward in Mundaka, in agreement with the location of major point sources of sewage. Labile material, proteins and protein:carbohydrate ratio showed upward increases in both estuaries, attributed to the oxygen restrictive conditions to decompose organic materials. Vertical distribution of organic components into sediments reflected the history of human activities. In Bilbao, the significant reduction of organic and labile compounds, and the decrease of protein:carbohydrate values in top layers seem a result of recent remedial actions to reduce urban sewage inputs and improve environmental conditions. Higher contributions of proteins and lipids were associated with anthropogenic materials, and higher contributions of carbohydrates with autochthonous materials. High protein:carbohydrate values indicate poorly decomposed materials of human origin. The close relationship of carbohydrates with chloropigments in Mundaka suggests that phytobenthic populations and derived detritus contributed to a greater extent to the organic fraction in this estuary.

Ubiquitination of Keap1, a BTB-Kelch Substrate Adaptor Protein for Cul3, Targets Keap1 for Degradation by a Proteasome-independent Pathway

Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.

HSP90: A Rising Star on the Horizon of Anticancer Targets

Over the past decade, heat-shock protein (HSP)90 has begun to draw increasing attention as a novel anticancer target with unique features. As a molecular chaperone, HSP90 promotes the maturation and maintains the stability of a large number of conformationally labile client proteins, most of which are involved in biologic processes that are often deranged within tumor cells, such as signal transduction, cell-cycle progression and apoptosis. As a result, and in contrast to other molecular targeted therapeutics, inhibitors of HSP90 achieve their promising anticancer activity through simultaneous disruption of many oncogenic substrates within cancer cells. This review provides a brief summary of HSP90 biology and its association with cancer. It describes the discovery and development of HSP90 inhibitors as anticancer agents and their current status in the clinic. Finally, it closes with a discussion of the unique challenges confronting the further development of these agents and their prospects for the future.

Increasing Expression of Heme Oxygenase-1 by Proteasome Inhibition Protects Astrocytes from Heme-mediated Oxidative Injury

Hemin is released from hemoglobin after CNS hemorrhage, and may contribute to cell loss in surrounding tissue. Heme oxygenase-1 (HO-1) is induced by these injuries, and may have an effect on cell viability. In a prior study, we reported that increasing HO-1 expression by adenoviral gene transfer prior to hemin exposure attenuated oxidative stress and cell death in astrocytes. However, rapid gene transfer to the CNS may not be feasible. HO-1 expression is controlled by a stress-responsive transcription factor, Nrf2, which is a labile protein that is subject to proteasomal degradation. In this study, we hypothesized that preventing degradation of Nrf2 with a lipid-soluble proteasome inhibitor would increase HO-1 expression and protect astrocytes from hemin. Treatment of cortical astrocyte cultures with 1 microM MG-132 resulted in a rapid increase in Nrf2, to a level that was five-fold that of vehicle-treated cultures by 2 h. This was followed by a three to six-fold increase in HO-1 expression that persisted through the 16 h observation period. Exposure of cultures to 30 microM or 60 microM hemin for 8 h resulted in death, as measured by LDH release, of 39+/-3.0 or 67.5+/-5.9% of astrocytes. Pre-treatment with MG-132 prevented approximately half of this injury. Cytoprotection persisted at 24 h, and was also observed when injury was assessed via the MTT assay. Astrocyte protein oxidation produced by hemin was also significantly attenuated by MG-132 pre-treatment. These results suggest that increasing HO-1 expression with a proteasome inhibitor protects astrocytes from heme-mediated oxidative injury. This pharmacological approach may provide a mechanism for rapidly upregulating HO-1 in astrocytes after CNS hemorrhage.

Water–Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T2' -selective 1H-13C-13C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T2' selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on 13C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones

We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.

Distinct IκB kinase regulation in adult T cell leukemia and HTLV-I-transformed cells

We have recently shown constitutive IκB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.

Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability

The intercoding regions between many Leishmania sp. genes regulate their mRNA expression. The MSPL mRNA, encoding a subclass of the major surface protease (MSP) of Leishmania chagasi, increases in abundance, when protein synthesis is arrested, while α-tubulin (α-TUB) mRNA and most other mRNAs do not. We found that the intercoding region between MSPL-coding regions, when cloned downstream of the β-galactosidase reporter gene ( β- GAL), caused β- GAL mRNA to increase 8- to 10-fold after inhibiting protein synthesis with cycloheximide. Stable L. chagasi transfectants containing hybrid MSPL/ α- TUB intercoding regions cloned downstream of β-GAL were made. The α-TUB intercoding region induced high-level baseline β- GAL mRNA that increased only 1.3-fold after incubation with cycloheximide. In contrast, the MSPL intercoding region, as well as constructs containing nucleotides 303–505 from the MSPL 3′UTR, caused steady-state β- GAL mRNA levels in the absence of cycloheximide that were approximately 10% of α-TUB constructs. These levels increased between 4.4- and 13.2-fold after cycloheximide was added. Constructs containing half of this region (303–394 or 395–505) produced intermediate levels of β-GAL mRNA and intermediate levels of cycloheximide induction. The kinetics of cycloheximide induction of β-GAL mRNA was similar with region 303–505 constructs as with constructs bearing the entire endogenous MSPL intercoding region. Furthermore, region 303–505 increased reporter mRNA abundance after cycloheximide by increasing mRNA half-life. Hence, we have identified a 202-nucleotide region within the MSPL 3′UTR that is in part responsible for cycloheximide induction. We hypothesize that this region may interact with labile regulatory protein factor(s).

The Importance of Having Thermosensor Control in the DnaK Chaperone System

In addition to the sigma(32)-mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADP-liganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40 degrees C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.

Preheating Accelerates Mitogen-activated Protein (MAP) Kinase Inactivation Post-heat Shock via a Heat Shock Protein 70-mediated Increase in Phosphorylated MAP Kinase Phosphatase-1

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MKP-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1.

Effect of EPS1 gene deletion in Saccharomyces cerevisiae on the secretion of foreign proteins which have disulfide bridges

Both amyloid-prone cystatin and unstable mutant C94A lysozyme were secreted in wild-type and Δeps1 Saccharomyces cerevisiae cells. Amyloid-prone cystatin secreted at much higher level in Δeps1 cells than that in wild-type yeast. In parallel, the secretion amount of disulfide bond disrupted mutant C94A lysozyme greatly increased in Δeps1 cells although that was apparently low in wild-type yeast cells compared with the secretion amount of wild-type lysozyme. It is interesting that neither the unstable mutant C94A lysozyme nor amyloid-prone cystatin secreted in Δeps1 cells maintained their specific activities. These observations lead to the supposition that yeast cells deficient for the protein disulfide isomerase-family-member EPS1 locus secrete more of labile disulfide-containing model proteins.

20S Proteasomal Degradation of Ornithine Decarboxylase Is Regulated by NQO1

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a very labile protein. ODC is a homodimeric enzyme that undergoes ubiquitin-independent proteasomal degradation via direct interaction with antizyme, a polyamine-induced protein. Binding of antizyme promotes the dissociation of ODC homodimers and marks ODC for degradation by the 26S proteasomes. We describe here an alternative pathway for ODC degradation that is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). We show that NQO1 binds and stabilizes ODC. Dicoumarol, an inhibitor of NQO1, dissociates ODC-NQO1 interaction and enhances ubiquitin-independent ODC proteasomal degradation. We further show that dicoumarol sensitizes ODC monomers to proteasomal degradation in an antizyme-independent manner. This process of NQO1-regulated ODC degradation was recapitulated in vitro by using purified 20S proteasomes. Finally, we show that the regulation of ODC stability by NQO1 is especially prominent under oxidative stress. Our findings assign to NQO1 a role in regulating ubiquitin-independent degradation of ODC by the 20S proteasomes.