Terrence M. Donohue

Enhancement of acetaldehyde−protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.

Ethanol consumption reduces the proteolytic capacity and protease activities of hepatic lysosomes

Chronic ethanol consumption causes decreased hepatic protein degradation, resulting in protein accumulation within hepatocytes. In this investigation, we sought to determine whether chronic ethanol feeding alters the degradative capacity and protease activities of isolated hepatic lysosomes. Male Sprague-Dawley-derived rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1-5 wk. Hepatic lysosomes were isolated by differential centrifugation and purified through Percoll gradients. Lysosomes obtained from livers of ethanol-fed rats degraded both endogenous protein substrates and the exogenously added radioactive substrate, 125I-RNase A, 26-42% more slowly than lysosomes from pair fed controls. The ethanol-elicited reduction in proteolytic capacity appeared to result in part, from a deficiency of the lysosomal cathepsins B, L, and H. Compared with controls, the specific activities of these enzymes were 31-45% lower in lysosomes from ethanol-fed rats. Immunoblot analyses also revealed that the intralysosomal as well as the intracellular content of cathepsin B was significantly lower in ethanol-fed rats. In contrast, ethanol consumption did not affect the cellular quantity of cathepsin L but lowered its amount in isolated lysosomes. Our findings suggest that chronic ethanol consumption causes a deficiency in lysosomal cathepsins by altering their biosynthesis and/or their trafficking into lysosomes.

Ethanol consumption alters trafficking of lysosomal enzymes and affects the processing of procathepsin L in rat liver.

In order to determine whether ethanol consumption alters the targeting of hepatic lysosomal enzymes to their organelles, we examined the sedimentation properties of lysosomal hydrolases in ethanol-fed rats and their pair-fed controls. Rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose dextrin for one to five wk. Liver extracts were fractionated by Percoll density gradient centrifugation and fractions obtained were analyzed for the distribution of lysosomal marker enzymes. Heavy lysosomes were further purified from these gradients and the activity of specific hydrolases was determined. Compared with those from controls, isolated lysosomes from ethanol-fed rats showed a 20-50% reduction in the activity of lysosomal acid phosphatase and beta-galactosidase. Decreased intralysosomal hydrolase activity in ethanol-fed rats was associated with a significant redistribution of these enzymes as well as those of cathepsins B and L to lighter fractions of Percoll density gradients. This indicated an ethanol-elicited shift of these enzymes to lower density cellular compartments. In order to determine whether ethanol administration affects the synthesis and proteolytic maturation of hepatic procathepsin L, we conducted immunoblot analyses to quantify the steady-state levels of precursor and mature forms of cathepsin L in hepatic post-nuclear fractions. Ethanol administration caused a significant elevation in the steady-state level of the 39 kDa cathepsin L precursor relative to its 30 kDa intermediate and 25 kDa mature product. These results were confirmed by pulse-chase experiments using isolated hepatocytes exposed to [35S]methionine. Hepatocytes from both control and ethanol-fed rats incorporated equal levels of radioactivity into procathepsin L. However, during the chase period, the ratios of the 39 kDa procathepsin L to its 30 kDa intermediate and 25 kDa mature product in cells from ethanol-fed rats were 1.5-3-fold higher than those in controls. These results demonstrate that ethanol consumption caused a marked impairment in the processing of procathepsin L to mature enzyme, without affecting its synthesis. Taken together, our findings suggest that chronic ethanol consumption caused a deficiency in intralysosomal enzyme content by altering the trafficking and processing of these hydrolases into lysosomes.

Glucose-6-phosphate dehydrogenase. Translational regulation of synthesis and regulation of processing of the enzyme in the uterus by estradiol

Abstract 12 h after intravenous administration of estradiol to ovariectomized mature rats, and 18-fold increase in the rate of synthesis of glucose-6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADP + oxidoreductase, EC 1.1.1.49) is observed. At that time, functional mRNA pools coding for this protein are only elevated 7-fold, suggesting that estradiol may be exerting effects at a posttranscriptional level of protein synthesis. The effect of estradiol on the rate of translation of the mRNA for this enzyme was evaluated by estimating the average ribosomal transit times in control and estradiol-treated rats under in vivo conditions. Transit times for glucose-6-phosphate dehydrogenase were decreased from 4.9 min in estrogen-deprived rats to 2.0 and 3.4 min, respectively, in 12 or 24 h estradiol-treated rats. Transit times for total released proteins in the cytosol were similarly decreased from 2.3 min to 1.0 and 1.3 min in the same groups of animals. Glucose-6-phosphate dehydrogenase has pyroglutamate as its NH 2 -terminus indicating that this region of the protein exists in a transient precursor form. The difference between the expected time after [ 3 H]glutamate administration that [ 3 H]pyroglutamate should be found in the enzyme and the observed first occurrence of [ 3 H]pyroglutamate in the enzyme is 9, 2.5 and 4 min in control and 12 or 24 h in estradiol-treated rats, respectively. This indicates that the ‘processing’ or removal of an NH 2 -terminal peptide from a percursor form of this enzyme is finalized after release of a glucose-6-phosphate dehydrogenase precursor from ribosomes and that the processing event is enhanced by estradiol.

Modulation of lysozyme function and degradation after nitration with peroxynitrite

BACKGROUND Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the proteins susceptibility to proteasome-catalyzed degradation. METHODS Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed. RESULTS Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein. CONCLUSION Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis. GENERAL SIGNIFICANCE Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.

Changes in the pathogenesis of alcohol-induced liver disease -- preclinical studies.

Excessive alcohol consumption presents considerable health risks in humans. A variety of morphologic and functional changes contribute to hepatic injury produced by heavy drinking. The present review summarizes the current knowledge of alcohol-induced liver disease and describes preclinical experimental approaches used to understand alcoholic liver disease (ALD), with a particular emphasis on impaired protein and lipid trafficking, disruption of proteolysis and autophagy, alterations in methionine metabolism and perturbations in metabolic signaling that cause dysfunctional gene expression and the eventual formation of aggresomal Mallory-Denk bodies (MDB) in liver cells. These changes eventually lead to some of the more severe hepatic impairments, including alcoholic hepatitis and fibrosis. Moreover the misuse of alcohol contributes to immune dysfunction and inadequate immune response to viral infections.

Glucose-6-phosphate dehydrogenase Partial characterization of the rat liver and uterine enzymes

Some properties of rat liver and uterine glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) have been determined. A procedure has been used for the purification of rat liver glucose-6-phosphate dehydrogenase to homogeneity (spec. act. 210-225 units/mg protein) from large amounts of liver (0.5-2 kg) with yields of up to 30%. Uterine glucose-6-phosphate dehydrogenase was obtained by immunoprecipitation methods and the properties of radioactively-labeled forms of this enzyme were then determined. The amino acid composition of the liver enzyme was found to be similar to that for the enzyme from other mammalian tissues. The liver and uterine enzymes have a subunit molecular weight of 57000 and a pI of 6.5. The NH2-terminal amino acid of both enzymes was found to be pyroglutamate.

Regulation of the Levels of mRNA for Glucose-6-phosphate Dehydrogenase and Its Rate of Translation in the Uterus by Estradiol

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyzes the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconolactone with the simultaneous reduction of NADP+ to NADPH. The enzyme is the first in the pentose phosphate pathway (hexose monophosphate shunt), which produces pentose phosphates used for nucleotide and nucleic acid synthesis, and reducing equivalents in the form of NADPH for use in biosynthetic reduction reactions, including fatty acid synthesis and hydroxlations. G6PD has been purified and characterized from the tissue cytosols of several mammalian species, including human erythrocytes, rat mammary gland, rat and mouse liver, and bovine adrenal cortex (Bonsignore and DeFlora, 1972; Levy, 1979). In general, the enzymes from these sources are “similar” and are composed of subunits that, by immunological and molecular weight criteria, may be identical proteins within a species. The monomeric subunits have a molecular weight of about 60,000, based on their rate of migration on SDS-polyacrylamide gels. The monomers are catalytically inactive. In the presence of NADP+, the inactive monomers form a catalytically active dimer containing 1 or perhaps 2 moles of NADP+ per mole of dimer; at reduced ionic strength and pH, and in the presence of Mg2+, the dimers aggregate to form tetramers and hexamers.

Rev-erb agonist and TGF-β similarly affect autophagy but differentially regulate hepatic stellate cell fibrogenic phenotype.

We demonstrated that ligand-activated nuclear receptor Rev-erbα mitigates CCl4-induced liver fibrosis. Rev-erbα is also a novel regulator of autophagy, a crucial eukaryotic catabolic system in which lysosomes degrade substrates for energy generation. In hepatic stellate cells (HSC) autophagy is reportedly required for this purpose to activate HSCs during fibrogenesis. Here, we examined whether pharmacological activation of Rev-erb with its synthetic ligand SR9009 or treatment with the pro-fibrotic cytokine, TGF-β, each differentially modulate autophagy to regulate the HSC phenotype. We measured the effects of SR9009 on autophagy markers in a CCl4-induced liver fibrosis model. Using primary and immortalized HSCs in vitro, we quantified SR9009 and TGF-β effects on autophagy flux. Compared with vehicle-treated controls, livers from CCl4-treated mice exhibited lower AMPK, higher P70S6K phosphorylation, elevated P62 and lower levels of ATG proteins, indicating a disruption of autophagosome (AV) formation. SR9009 treatment prevented CCl4-induced P70S6K phosphorylation but did not affect CCl4-induced changes in AMPK, ATG proteins or P62. Analysis of autophagy markers and autophagy flux in primary HSCs or an immortalized human HSC line (LX2), revealed that SR9009 exposure down-regulated AV biogenesis. These events were associated with lower levels of fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation. However, HSC exposure to TGF-β enhanced fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation, while it simultaneously decelerated AV synthesis. The autophagy activator rapamycin and the autophagy inhibitor wortmannin each decreased HSC activation, P70S6K phosphorylation and HSC proliferation. Furthermore, knock-down of P70S6K using siRNA blocked basal and TGF-β-induced cell proliferation in human activated LX2. We conclude that SR9009 and TGF-β both similarly affected autophagy but, differentially regulated HSC fibrogenic phenotype through modulation of P70S6K, which is crucial for cell proliferation and fibrogenesis.

The increase in hepatic tyrosine aminotransferase activity by ethanol administration involves an acceleration of enzyme synthesis

The present study was conducted to examine the nature of the increase in tyrosine aminotransferase (TAT) activity by acute ethanol administration. A significant rise in aminotransferase activity was observed as early as 1 hr after intact rats were gavaged with ethanol. Ethanol administration also increased TAT activity in adrenalectomized rats. Inhibition of ethanol metabolism by pyrazole administration had no effect on the ethanol-induced increase in TAT activity. Immunochemical analyses revealed that the enhancement of TAT activity in ethanol-fed rats correlated with an increase in aminotransferase protein. Measurement of the rate of TAT synthesis showed that in ethanol-fed rats, [3H]leucine was incorporated into the aminotransferase protein at a higher rate than in controls by a factor which was similar to the enhancement in enzyme activity. Our findings indicate that an acceleration of TAT synthesis fully accounts for the increase in TAT activity during the early stage of enzyme induction. TAT induction by ethanol administration is not dependent upon an increase in adrenal corticosteroid production, nor does it require ethanol metabolism.