Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935.

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.

Rat sinusoidal liver endothelial cells (SECs) produce pro-fibrotic factors in response to adducts formed from the metabolites of ethanol.

Previous studies with alcohol-associated malondialdehyde-acetaldehyde (MAA)-modified proteins have demonstrated an increase in the expression of adhesion molecules, and the secretion of pro-inflammatory cytokines/chemokines by rat sinusoidal liver endothelial cells (SECs). However, no studies have been initiated to examine the effects of MAA-modified proteins on the expression of the extracellular matrix (ECM) protein, fibronectin and its isoforms. For these studies, SECs were isolated from the liver of normal rats, and exposed to MAA-modified bovine serum albumin (MAA-Alb). At selected time points, the total plasma and cellular fibronectin were determined by Western blot. Injection of rat liver via the mesenteric vein with MAA-Alb was performed in an effort to evaluate the potential in vivo role of MAA-modified proteins in the development of fibrosis. Expression of both plasma and cellular fibronectin was significantly increased over controls in the MAA-Alb stimulated SECs (>3-fold). Importantly, the isotype of fibronectin secreted was determined to be of the EIIIA variant and not EIIIB. These data were confirmed using RT-PCR procedures on liver tissue from; isolated SECs, and from an in vivo animal model wherein MAA-Alb was administered via the mesenteric vein. Thus, these studies demonstrate that MAA-modified proteins initiate a pro-fibrogenic response by initiating the expression of the fibronectin EIIIA isoform by SECs.

Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins.

Scavenger receptors on sinusoidal liver endothelial cells (SECs) eliminate potentially harmful modified proteins circulating through the liver. It was shown recently that aldehyde-modified proteins bind to scavenger receptors and are associated with the development/progression of alcoholic liver diseases. For these studies, rat livers were perfused in situ with 125I-formaldehyde-bovine serum albumin (f-Alb) or 125I-malondialdehyde-acetaldehyde-bovine serum albumin (MAA-Alb) in the presence of known scavenger receptor ligands as inhibitors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and scavenger receptor Type A (SRA) knock-out mice were used to assess the role of these receptors in mediating immune responses. The degradation of 125I-f-Alb or 125I-MAA-Alb in whole livers and isolated SECs can be inhibited by known scavenger receptor ligands, including f-Alb, maleylated bovine albumin, and fucoidan. 125I-f-Alb could not be completely inhibited by MAA-Alb. In contrast, 125I-MAA-Alb was only partially inhibited with advanced glycosylated endproduct albumin. RT-PCR data show the presence of a number of scavenger receptors on SECs that may be responsible for the binding of MAA-modified proteins. SRA seems to be one of these receptors involved in the effects mediated by MAA-modified proteins. In a study using SRA knockout mice, it was shown that a decreased antibody response to MAA-Alb resulted. By RT-PCR, CD36, LOX-1, and SR-AI are the scavenger receptors most likely involved in the degradation of MAA-Alb.

Aldehydes in cigarette smoke react with the lipid peroxidation product malonaldehyde to form fluorescent protein adducts on lysines.

Cigarette smoke is a risk factor for the development of several diseases, but the exact mechanism responsible has not been well-characterized. Because modification, or adducting, of biomolecules is thought to mediate the toxic effects observed from exposure to a wide variety of harmful chemicals, this study investigated the ability of cigarette smoke to produce specific adducts on a peptide to gain insight into the likely effect on cellular proteins. We describe the modification of the epsilon-amino group of lysine contained in a test peptide with stable fluorescent adducts derived from monofunctional aldehydes occurring in cigarette smoke and malonaldehyde, a product of lipid peroxidation. Utilizing high-performance liquid chromatography, fluorescent measurements, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, the 1,4-dihydropyridine-3,5-dicarbaldehyde and 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivatives of lysine were identified as products of exposure to cigarette smoke extract and malonaldehyde. These data suggest that cigarette smoke may promote the modification of proteins, like those associated with oxidized low-density lipoprotein, and may contribute to smoking-related disease.

Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells.

BACKGROUND: The possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury. METHODS: We have recently demonstrated that the MAA-adduct induces tumor necrosis factor-alpha (TNF-alpha) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-alpha were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-alpha concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats. CONCLUSION: These results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-alpha release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-alpha release measured following both MAA-Alb and LPS stimulation.

Lipopolysaccharide is a cofactor for malondialdehyde-acetaldehyde adduct-mediated cytokine/chemokine release by rat sinusoidal liver endothelial and Kupffer cells.

BACKGROUND: The nonparenchymal cells of the liver have been suggested to play a significant role in the inflammatory processes observed in the development and/or progression of alcoholic liver disease. Our laboratories have shown that malondialdehyde-acetaldehyde (MAA)-modified proteins can induce immune responses, cytokine/chemokine secretion, and antigen processing and presentation by liver sinusoidal endothelial cells (SECs). Another molecule that has been shown to induce similar types of responses in Kupffer cells (KCs) is lipopolysaccharide (LPS). Because these materials induce similar responses, it was the purpose of this study to investigate the relationship between LPS and MAA-modified proteins in the development of proinflammatory responses by SECs and KCs. METHODS: For these studies, SECs and KCs were isolated from chow-fed, pair-fed, and ethanol-fed rats. Cells were stimulated with media alone, bovine serum albumin (Alb), or MAA-modified Alb (MAA-Alb) in the presence or absence of LPS 1 ng/ml, and the supernatants were assayed by enzyme-linked immunosorbent assay for tumor necrosis factor alpha, macrophage chemotactic protein 1, and macrophage inhibitory protein. RESULTS: All three cytokines/chemokines were 3 to 5 times higher when SECs or KCs were stimulated by MAA-Alb in the presence of LPS, in contrast to cells stimulated with Alb or media in the presence of LPS. Chronic ethanol consumption (6 weeks) had variable effects on the secretion of these cytokines/chemokines but in general did not alter the increased secretion in response to MAA-Alb in the presence of LPS. CONCLUSIONS: These studies strongly suggest that the sensitization of SECs and KCs by LPS plays a significant role in the development and/or progression of alcoholic liver disease, and the subsequent activation by MAA-modified proteins may be a mechanism by which proinflammatory processes are initiated.

Immunologic mechanisms of alcoholic liver injury.

Clinically, the association of alcoholic liver disease (ALD) with circulating autoantibodies, hypergammaglobulinemia, antibodies to unique hepatic proteins, and cytotoxic lymphocytes reacting against autologous hepatocytes strongly suggests altered immune regulation with an increased activity toward normal self-proteins (loss of tolerance). Experimentally, there are several immune responses generated specifically recognizing self-proteins that are modified by metabolites of alcohol. These data strongly suggest that immune reactions may play a significant role in inducing and sustaining an inflammatory cascade of tissue damage to the liver. Additional support for this comes from the observation that the histological appearance of livers with ALD is that of a chronic active hepatitis-like inflammatory disease. Therefore, the hypothesis that immune mechanisms are involved in recurrent alcoholic hepatitis, although not summarily proven, is reasonable, supported by clinical and experimental evidence, and the subject of this article.

Mechanisms of alcohol liver damage: aldehydes, scavenger receptors, and autoimmunity.

While most of the investigations into the causative events in the development of alcoholic liver disease (ALD) have been focused on multiple factors, increasing interest has centered around the possible role of immune mechanisms in the pathogenesis and perpetuation of ALD. This is because many of the clinical features of ALD suggest that immune effector mechanisms may be contributing to liver tissue damage, as evidenced by the detection of circulating autoantibodies, and the presence of CD4+ and CD8+ lymphoid cells in the livers of patients with ALD. One mechanism that has been associated with the development of autoimmune responses is the modification (haptenation or adduction) of liver proteins with aldehydes or other products of oxidative stress. This is because it has been shown that these adducted proteins can induce specific immune responses, to the adduct, the adduct plus protein (conformational antigens), as well as the unmodified parts of the protein. More importantly, it is possible to demonstrate that adducted self-proteins can induce reactivity to the normal self-protein and thereby induce autoimmune responses. Therefore, it is the purpose of this manuscript to outline the mechanism(s) by which these modified self proteins can induce autoimmune reactivity, and thus play a role in the development and/or progression of ALD.

Chronic ethanol consumption impairs receptor-mediated endocytosis of MAA-modified albumin by liver endothelial cells.

Alcoholic liver disease has been associated with abnormalities in receptor-mediated endocytosis (RME) which results in abnormal degradation of metabolically altered proteins. Model systems using formaldehyde-modified albumin (f-Alb) have shown an impairment in RME following chronic alcohol consumption utilizing both in situ perfused rat livers and isolated rat liver endothelial cells (LECs). The discovery that alcohol metabolite derived aldehydes can modify proteins prompted a study to determine if malondialdehyde-acetaldehyde-modified albumin (MAA-Alb) would be degraded similar to that reported for f-Alb, and whether ethanol-fed rats would demonstrate an impaired RME with respect to this ligand which occurs as a consequence of chronic ethanol consumption. MAA-Alb was degraded slightly more than f-Alb in both in situ perfused livers and at the single cell level. This degradation was completely inhibited with 100x unlabeled f-Alb, which suggests the use of a similar receptor. Following alcohol consumption there was a 50-60% decrease in MAA-Alb degradation in whole livers and isolated LECs. Utilizing isolated LECs it was determined that impairment in internalization was the most likely mechanism for the decrease in the amount of MAA-Alb that was degraded. These data show that chronic alcohol consumption by rats does in fact impair RME of alcohol metabolite-derived adducted proteins, and this impairment is due to a defect in the post-internalization step rather than the binding or degradation of the modified protein.

ATA2-mediated amino acid uptake following partial hepatectomy is regulated by redistribution to the plasma membrane.

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.

Inhibition of hepatic stellate cell collagen synthesis by N-(methylamino)isobutyric acid.

The increased deposition of extracellular matrix by hepatic stellate cells following liver injury, in a process known as activation, is considered a key mechanism for increased collagen content of liver during the development of liver fibrosis. We report that N-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of System A-mediated amino acid uptake, reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a rat model of liver injury and fibrosis. Rat CFSC-2G cells were cultured in 0-5mM MeAIB, and the accumulation and synthesis of collagen were measured by binding to Sirius red F3B and pulse-labeling with [3H]-proline, respectively. The effect of MeAIB on collagen accumulation in vivo was evaluated utilizing a rat model of hepatic fibrosis. MeAIB inhibited collagen accumulation in CFSC-2G cultures in a concentration-dependent manner with 5mM MeAIB reducing collagen 44.6+/-1.2% compared with the control. In CFSC-2G cultures, MeAIB selectively inhibited the incorporation of proline into cellular macromolecules by 43+/-4%, while the synthesis of proteins containing leucine was not affected. In vivo, oral administration of 160mg MeAIB/kg body weight per day to rats significantly reduced the hepatic collagen accumulation in response to 1 week of CCl(4)-induced liver injury. MeAIB reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a CCl(4)-induced rat model of liver injury and fibrosis.

Phosphoenolpyruvate carboxykinase is induced in growth-arrested hepatoma cells.

Phosphoenolpyruvate carboxykinase (PEPCK) mRNA is elevated in H4IIEC3 rat hepatoma cells cultured at high density, suggesting that PEPCK expression and growth arrest may be coordinately regulated. Induction of growth arrest either by contact inhibition (high culture density) or by serum deprivation correlated with significant increases in PEPCK protein and its mRNA. The observation that PEPCK mRNA was induced by contact inhibition in the presence of serum indicates that the effect of high density is independent of insulin or any other serum component. The magnitudes of the changes in PEPCK expression during growth arrest were greatly enhanced in KRC-7 cells, an H4IIEC3 subclone that is much more sensitive to growth arrest than its parental cell line. Restimulation of proliferation in growth-arrested KRC-7 cells, either by addition of serum or insulin to serum-deprived cells or by replating contact-inhibited cells at low density, caused a rapid decrease in PEPCK expression. However, PEPCK mRNA is not always reduced in proliferating cells since treatment of serum-starved cells with epidermal growth factor stimulated entry into the cell cycle but did not affect PEPCK mRNA levels. Finally, dexamethasone induction of PEPCK mRNA was blunted in cells cultured at high density but was unaffected by the presence or absence of serum. Collectively, these data suggest the possibility of cross-talk between the control of PEPCK expression and growth arrest in KRC-7 cells.