NADPH-dependent microsomal electron transfer increases degradation of CYP2E1 by the proteasome complex: role of reactive oxygen species.

Increased levels of cytochrome P450 2E1 (CYP2E1) produced by low-molecular-weight compounds is mostly due to stabilization of the enzyme against proteolytic degradation. CYP2E1, in the absence of substrate or ligand, normally has a short half-life, but the factors which regulate CYP2E1 turnover or trigger its rapid degradation are not known. Since CYP2E1 is active in producing reactive oxygen species, experiments were carried out to evaluate whether reactive oxygen species modulated the degradation of CYP2E1. CYP2E1 present in human liver microsomes was very stable. Addition of the cytosol fraction produced degradation of CYP2E1, and this was enhanced when NADPH was present in the reaction system. Antioxidants or iron chelators which prevent lipid peroxidation, prevented the degradation of CYP2E1 by the cytosolic fraction. Similarly, diphenyleneiodonium chloride, which inhibits NADPH-dependent electron transfer, prevented the degradation of CYP2E1, as did 4-methylpyrazole, a ligand which increases the level of CYP2E1. If microsomes were first incubated with NADPH for 30 min, followed by the addition of these agents, there was no protection against CYP2E1 degradation. Lactacystin, an inhibitor of the proteasome, decreased the degradation of CYP2E1. In intact HepG2 cells transduced to express CYP2E1, proteasome inhibitors elevated steady-state levels of CYP2E1. Steady-state levels of CYP2E1 were increased by about 50% when the cells were incubated with trolox. Trolox decreased the rate of loss of CYP2E1 protein when the cells were treated with cycloheximide. These results suggest that NADPH-dependent production of reactive oxygen species may result in oxidative modification of CYP2E1, followed by rapid degradation of the labilized CYP2E1 by the proteasome complex. It is interesting to speculate that one consequence of the high rates of production of reactive oxygen species by CYP2E1 is its own labilization and subsequent rapid degradation, and this may be a regulatory mechanism to prevent high levels of the enzyme from accumulating within the cell.     

Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1-catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1-dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1-dependent O-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.     

The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum.

The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.     

Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins

We have characterized a pre-Golgi, proteolytic pathway for rapid degradation of newly synthesized T cell receptor (TCR) subunits which is insensitive to drugs that block lysosomal proteolysis. The site of degradation in this pathway is either part of or closely related to the endoplasmic reticulum (ER). This "ER" degradative pathway very likely plays an important role in many cells in the removal of unassembled or incompletely assembled membrane protein complexes from the secretory pathway. It is the sole pathway followed by TCR alpha chains and alpha-beta complexes in transfected fibroblasts. In T cells treated with ionophores, which disrupt transport of the TCR from the ER to the Golgi, all newly synthesized alpha, beta, and delta chains are destroyed by this pathway. A variety of biochemical and morphological techniques have been used to distinguish the "ER" degradative pathway from an alternative, lysosomal pathway.     

Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase-mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.     

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.     

Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+)

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.     

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.     

Proteolysis in homogenates of perfused rat liver: responses to insulin, glucagon and amino acids

The proteolytic release of leucine and isoleucine was assessed in homogenates of rat livers perfused under conditions known to influence protein degradation in the intact liver. Release was increased by perfusion alone and by additions of glucagon and was inhibited by insulin and amino acids. These responses correlated both with rates of proteolysis during perfusion and with physical alterations of the lysosomal system, reported earlier. Homogenate proteolysis appeared to comprise two components: the release of free amino acids from the total particulate fraction and from peptides in the cytosol. Both components are believed to be generated by elements of the lysosomal system.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination

Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.     

NAADP mobilizes calcium from the endoplasmic reticular Ca(2+) store in T-lymphocytes

The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H(+)-ATPase, did not block or reduce NAADP-induced Ca(2+) signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394-21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum.     

Effect of organic solvents on catalytic and functional activity of transport Ca2+,Mg2+-ATPase from smooth muscle cell membrane

It was shown that organic solvents (dioxane, acetone, ethanol, dimethylsulfoxide) at concentrations of < 10% suppress the activity of transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membranes of smooth muscle cells and Mg(2+)-ATP-dependent accumulation of Ca2+ ions in inverted membrane vesicles. It was found that one of the reasons for the inhibition of enzymatic and transport activity of Ca2+, Mg(2+)-ATPase by the action of these solvents is an increase in the attractive force between oppositely charged active center of the enzyme and the product (products) of the ATP-hydrolase reaction, which is induced by a decrease in the dielectric permeability of incubation medium.     

Degradation of cytochrome P450 2E1: selective loss after labilization of the enzyme.

Mechanism-based inactivation of cytochrome P450 can result in the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. In the present study we took advantage of different modes of inactivation of P450 2E1 by CCl4, 1-aminobenzotriazole, or 3-amino-1,2,4-triazole to investigate parameters which target P450 2E1 for proteolysis from the microsomal membrane. Treatment of mice with CCl4 at the point of maximal induction of P450 2E1 after a single oral dose of acetone resulted in the complete loss of P450 2E1-dependent p-nitrophenol hydroxylation and a 75% loss of immunochemically detectable protein within 1 h of administration. Treatment with 1-aminobenzotriazole at the point of maximal induction caused a complete loss of P450 2E1-dependent p-nitrophenol hydroxylation but only a 12% loss of immunochemically detectable protein 1 h after administration. Treatment of mice with 3-amino-1,2,4-triazole caused a rapid loss of both catalytic activity and microsomal p-nitrophenol hydroxylase activity. However, unlike CCl4 treatment, the activity and enzyme level rebounded 5 and 9 h after treatment. The P450 2E1 ligand, 4-methylpyrazole, administered at the point of maximal induction maintained the acetone-induced catalytic and immunochemical level of P450 2E1. These results suggest that differentially modified forms of P450 2E1 show a characteristic susceptibility to degradation. While there are many potential pathways for protein degradation, the loss of P450 2E1 was associated with increased formation of high molecular weight microsomal ubiquitin conjugates. The formation of ubiquitin-conjugated microsomal protein which correlates with P450 2E1 loss suggests that ubiquitination may represent a proteolytic signal for the rapid and selective proteolysis of certain labilized conformations of P450 2E1 from the endoplasmic reticulum.     

Two distinct proteolytic systems responsible for glucose-induced degradation of fructose-1,6-bisphosphatase and the Gal2p transporter in the yeast Saccharomyces cerevisiae share the same protein components of the glucose signaling pathway.

Addition of glucose to Saccharomyces cerevisiae inactivates the galactose transporter Gal2p and fructose-1,6-bisphosphatase (FBPase) by a mechanism called glucose- or catabolite-induced inactivation, which ultimately results in a degradation of both proteins. It is well established, however, that glucose induces internalization of Gal2p into the endocytotic pathway and its subsequent proteolysis in the vacuole, whereas FBPase is targeted to the 26 S proteasome for proteolysis under similar inactivation conditions. Here we report that two distinct proteolytic systems responsible for specific degradation of two conditionally short-lived protein targets, Gal2p and FBPase, utilize most (if not all) protein components of the same glucose sensing (signaling) pathway. Indeed, initiation of Gal2p and FBPase proteolysis appears to require rapid transport of those substrates of the Hxt transporters that are at least partially metabolized by hexokinase Hxk2p. Also, maltose transported via the maltose-specific transporter(s) generates an appropriate signal that culminates in the degradation of both proteins. In addition, Grr1p and Reg1p were found to play a role in transduction of the glucose signal for glucose-induced proteolysis of Gal2p and FBPase. Thus, one signaling pathway initiates two different proteolytic mechanisms of catabolite degradation, proteasomal proteolysis and endocytosis followed by lysosomal proteolysis.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.