Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway. (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins. (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2's) except E2(20K) and E2(230K). Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted.     

A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin

Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.     

Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.     

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.     

A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties

In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.     

Relationship Between the Ubiquitin-Dependent Pathway and Apoptosis in Different Cells of the Central Nervous System: Effect of Thyroid Hormones

We have recently shown that sustained neonatal hyperthyroidism in the rat activates apoptosis of oligodendroglial cells (OLGc) and that inhibition of the proteasome-ubiquitin (Ub) pathway by lactacystin produces increased apoptosis in cerebellar granule cells (CGC). In the present study we have analyzed the relationship between the activation of the Ub-dependent pathway, the expression of the Ub genes and programmed cell death in neurons of the rat cerebellum and cerebral cortex and in OLGc. This study was carried out in normal animals, in rats submitted to sustained neonatal hyperthyroidism and in cell cultures treated with an excess of thyroid hormones. In neurons of the cerebral cortex, thyroid hormone produces an increase of Ub-protein conjugates, an enhancement in the expression of the Ub genes and an increase in apoptosis, while the opposite results are obtained in CGC. These results indicate that in neurons, the changes in the cell death program produced by thyroid hormone run in parallel with those occurring in the Ub-dependent pathway. In OLGc, thyroid hormone increases apoptosis but does not produce changes in the Ub pathway. Preliminary studies indicate that in coincidence with what occurs in optic nerves, the sciatic nerves both in controls and in hyperthyroid animals are unable to form Ub-protein conjugates. These results indicate that in cells of the CNS such as neurons, in which the Ub-dependent pathway is actively expressed, it appears to be closely correlated with apoptosis.     

ATP-dependent degradation of 125I-bovine serum albumin by rabbit reticulocytes does not need repression of an endogenous inhibitor

The ubiquitin-dependent proteolysis of 125I-bovine serum albumin in rabbit reticulocytes has been investigated. Using various reticulocyte fractions (reticulocyte protease, inhibitor-free protease, "ubiquitin" and "inhibitor") in the presence or absence of ATP, we found that the repression of an endogenous inhibitor, as suggested by others for alpha-casein proteolysis, is unlikely for bovine serum albumin. Therefore, differences exist in the ATP-dependent proteolytic pathway of rabbit reticulocytes depending on the substrate. Fractionation of the reticulocyte ATP-dependent proteolytic system revealed at least two proteolytic and two inhibitory fractions involved in the proteolysis of bovine serum albumin.     

Cutting Edge: Selective role of ubiquitin in MHC class I antigen presentation

The importance of ubiquitination in MHC class I-restricted Ag processing remains unclear. To address this issue, we overexpressed wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells using an inducible vaccinia virus system. Overexpression of the lysineless Ub nearly abrogated polyubiquitination and potently inhibited epitope presentation from a cytosolic N-end rule substrate as well as endoplasmic reticulum (ER)-targeted model Ags. In contrast, there was little impact on Ag presentation from cytosolic proteins. These trends were location dependent; redirecting cytosolic Ag to the ER rendered presentation lysineless Ub-sensitive, whereas retargeting exocytic Ag to the cytosol had the inverse effect. This dichotomy was further underscored by small interfering RNA knockdown of the ER-associated Ub ligase Hrd1. Thus, Ub-dependent degradation appears to play a major role in the MHC class I-restricted processing of ER-targeted proteins and a more restricted role in the processing of cytosolic proteins.     

The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.     

Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells

Arsenic contamination is a principal environmental health threat throughout the world. However, little is known about the effect of arsenic on steroidogenesis in granulosa cells (GCs). We found that the treatment of preovulatory GCs with arsenite stimulated progesterone production. A significant increase in serum level of progesterone was observed in female Sprague-Dawley rats following arsenite treatment at a dose of 10 mg/L/rat/day for 7 days. Further experiments demonstrated that arsenite treatment did not change the level of intracellular cyclic AMP (cAMP) or phosphorylated ERK1/2 in preovulatory GCs; however, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. This implied that the effect of arsenite on progesterone production may require cAMP/PKA and ERK1/2 signaling but not depend on them. Furthermore, we found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH levels. Arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by arsenite or follicle-stimulating hormone (FSH). GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment. Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment.     

Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins

The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone.     

Identification of a ubiquitin- and ATP-dependent protein degradation pathway in rat cerebral cortex.

To investigate the existence of a ubiquitin-dependent protein degradation system in the brain, the proteolytic activity of the cerebral cortex was examined. The soluble extract of rat cerebral cortex degraded 125I-radiolabeled lysozyme in an ATP-dependent manner. The ATP-dependent proteolysis was suppressed with iodoacetamide, which inhibits ubiquitin conjugation, and was abolished by blocking of the amino residues of lysozyme. These results suggest the participation of ubiquitination in the proteolytic activity. An ATP-dependent 125I-ubiquitin-conjugating activity was detected in fraction II from the cerebral cortex. The presence of ATP-dependent proteolytic activity which acted preferentially on ubiquitinated lysozyme was demonstrated, using ubiquitin-125I-lysozyme conjugates as a substrate. The proteinase had a molecular mass of 1500 kDa and displayed nucleotide dependence and sensitivity to various proteinase inhibitors similar to those of the 26S proteinase complex found in reticulocytes. Dialysis of the soluble fraction caused a decrease in the proteolytic activity of ATP-dependent and preferential for ubiquitin-lysozyme conjugates and a reciprocal increase in the ATP-independent free 125I-lysozyme-degrading activity which was scarcely detected before dialysis. The former ATP-dependent proteolytic activity may play a physiological role in the brain.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 microM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67(phox) localized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.     

Arsenite activation of P13K/AKT cell survival pathway is mediated by p38 in cultured human keratinocytes.

BACKGROUND: Arsenic has been considered as a carcinogen. Recently the issue of arsenic in drinking water raised an unprecedented social concern on human health, and yet the molecular mechanisms through which arsenic induces cancer remain unknown. Activation of cell survival pathway leading to the activation of eNOS has been associated with various types of cancer. The objective of this study was to investigate the pathway leading to the activation of eNOS in response to arsenite using human keratinocytes. MATERIALS AND METHODS: Cultured keratinocytes (HaCat cells) were exposed to arsenite with or without pretreatment of various inhibitors. Western blot analysis was performed to determine the activation of p38, AKT, eNOS. EGFR tyrosine phosphorylation was detected by immunoprecipitation and Western blot analysis. pNPP assay was used to measure phosphatase activity in cell lysate. FACS analysis was performed for the determination of generation of reactive oxygen species. RESULTS: Arsenite induced the activation of AKT at both Ser473 and Thr308, and its downstream effector eNOS in cultured human keratinocytes. Arsenite also induced phosphorylation of p38. PI-3-kinase inhibitors, Wortmannin and LY294002 inhibited arsenite-induced phosphorylation of AKT and eNOS but had no effect on phosphorylation of p38. Interestingly, however, SB203580, a known p38 inhibitor, completely inhibited arsenite-induced phosphorylation of AKT and eNOS. Arsenite induced generation of reactive oxygen species and inactivated phosphatase activity, but did not activate EGF receptor tyrosine phosphorylation. CONCLUSIONS: Collectively, our data indicate that arsenite induces activation of AKT and eNOS, via PI-3-kinase and p38 pathway, likely bypassing the activation of EGF receptor in cultured human keratinocytes.     

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.     

Functional heterogeneity of ubiquitin carrier proteins

In the formation of covalent ubiquitin-protein conjugates that occurs during ATP- and ubiquitin-dependent proteolysis in reticulocyte extracts, ubiquitin (Ub) is activated to a thiol ester of the activating enzyme E1 (via the Ub carboxyl terminus), transferred to low-molecular weight "carrier proteins" (E2s) to form E2-Ub thiol esters, and then transferred by a third enzyme (E3) to amino groups on target proteins (Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214). We report here the fractionation of Ub carrier proteins by molecular weight, and their characterization with respect to several activities. The Ub thiol ester forms of at least four of the five E2s catalyze Ub transfer to a number of small amines, in a reaction that does not require E3; only primary amines on primary carbons can serve as Ub acceptors. E3-independent Ub transfer to the small, basic proteins histones H2A and H2B, and cytochrome c, is also observed. The Ub thiol ester forms of two of the E2s were found to catalyze Ub transfer to cytochrome c. Only a single E2 functions in E3-dependent conjugate formation (with the substrates creatine phosphokinase, reduced/carboxymethylated serum albumin, and oxidized RNase) and in E3-dependent protein breakdown (with the substrate serum albumin). This E2 has a subunit molecular weight of 14,000 and migrates as a dimer on Sephacryl 200.     

Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

26S proteasome regulatory particle mutants have increased oxidative stress tolerance

The 26S proteasome (26SP) is a multi-subunit, multi-catalytic protease that is responsible for most of the cytosolic and nuclear protein turnover. The 26SP is composed of two sub-particles, the 19S regulatory particle (RP) that binds and unfolds protein targets, and the 20S core particle (20SP) that degrades proteins into small peptides. Most 26SP targets are conjugated to a poly-ubiquitin (Ub) chain that serves as a degradation signal. However, some targets, such as oxidized proteins, do not require a poly-Ub tag for proteasomal degradation, and recent studies have shown that the main protease in this Ub-independent pathway is free 20SP. It is currently unknown how the ratio of 26SP- to 20SP-dependent proteolysis is controlled. Here we show that loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a leads to decreased 26SP accumulation, resulting in reduced rates of Ub-dependent proteolysis. In contrast, all three RP mutants have increased 20SP levels and thus enhanced Ub-independent protein degradation. As a consequence of this shift in proteolytic activity, mutant seedlings are hypersensitive to stresses that cause protein misfolding, and have increased tolerance to treatments that promote protein oxidation. Taken together, the data show that plant cells increase 20SP-dependent proteolysis when 26SP activity is impaired.