Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells

We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 wk elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by MS. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.     

Cytosolic insulin-binding proteins of mouse liver cells

It has been recently shown that insulin retains its biological activity after receptor-directed internalization and it may affect the cell metabolism by interaction with cytosolic insulin-binding proteins (CIBPs). Using affinity chromatography combined with SDS-PAGE and MALDI-TOF mass-spectrometry we have identified 7 proteins from mouse liver cells that specifically bind to the insulin, including adenylate kinase 2 (25.6 kD), kinesin superfamily protein 20B (26.0 kD), hepatic arginase 1 (34.8 kD), fructose-bisphosphate aldolase B (39.5 kD), 4-hydroxyphenylpyruvate dioxygenase (45.1 kD), betaine-homocysteine methyl-transferase (45.0 kD) and KRIT1 (83.4 kD).     

Persistent expression of hepatitis C virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.     

Identification of oxidized plasma proteins in Alzheimer's disease

The modification of proteins by reactive oxygen species is central to the pathology of Alzheimer's disease (AD). Previously, we have observed specific oxidized proteins in blood plasma of AD subjects [Biochem. Biophys. Res. Commun. 275 (2000) 678]. Plasma from AD subjects and age-matched controls was subjected to two-dimensional gel electrophoresis (2-DE). Oxidized proteins with new carbonyl groups were detected by reaction with 2,4-dinitrophenylhydrazine, followed by Western blotting with anti-DNP antibody. Seven principal oxidized protein spots (isoelectric point=4.7-5.5; molecular mass=45-65 kDa) were observed, with varying levels of oxidation in plasma samples from both AD and non-AD subjects. Matrix-assisted laser desorption mass spectroscopy (MALDI-TOF/MS) revealed that these oxidized proteins were isoforms of fibrinogen gamma-chain precursor protein and of alpha-1-antitrypsin precursor. These proteins exhibited a two- to sixfold greater specific oxidation index in plasma from AD subjects when compared to controls. Both these proteins have been previously implicated in the pathology of the disease. It is possible that oxidized isoforms of these proteins may serve as biomarkers for AD.     

Identification of carbohydrate-binding proteins from mouse and human fibroblasts.

Three carbohydrate-binding proteins (Mr 35 000, 16 000 and 13 500) were isolated from extracts of mouse 3T3 fibroblasts by affinity chromatography on polyacrylamide beads to which was covalently bound the ligand 6-aminohexyl 4-beta-D-galactosyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. None of these proteins bind to polyacrylamide beads coupled with either 6-aminohexanol or 6-aminohexyl beta-D-galactopyranoside. Therefore they appear to be carbohydrate-binding proteins specific for galactose-terminated glycoconjugates. A carbohydrate-binding protein was also purified from extracts of human foreskin fibroblasts. This protein (Mr 35000) may represent the human counterpart of the mouse protein of similar Mr and binding properties.     

Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures

The degradation rates of inner mitochondrial membrane proteins were examined in serum-deprived hepatoma cultures. In those nonproliferating cells, the degradation of composite mitochondrial proteins was a first order process with a half-life of 34 h. The half-lives of specific inner mitochondrial membrane polypeptides were determined by examining the 3H/35S of isolated polypeptides from cells given [3H]methionine and [35S]methionine pulses, respectively, before and after a 2-day chase period. The 33 most abundant polypeptides resolved on a bidirectional polyacrylamide gel system showed half-lives ranging from 20 to 100+ h. The 15 polypeptides translated on mitochondrial ribosomes in the presence of inhibitory concentrations of cycloheximide also displayed heterogeneous rates of degradation (t1/2 = 35-100+ h). None of the isolated adenosine triphosphatase (coupling factor F1) or immunoprecipitated cytochrome c oxidase subunits were significantly turned over during the case period. Five of eight cytochrome b-c1 complex subunits, however, were turned over significantly more rapidly (t1/2 = 39-42 h) than the other three (t1/2 = 94+ h). The results demonstrate heterogeneous degradation rates for inner membrane polypeptides, extending in some cases to those within the same respiratory complex.     

Stochastic esr analysis of rat liver and hepatoma mitochondrial lipids

A commonly used model for the interaction of the motional narrowing of ESR lines is shown to be qualitatively misleading. An analysis of lipid extracts of mitochondrial preparations labeled with 12-nitroxide stearic acid produced linear plots of the logarithm of the correlation time versus the reciprocal of the absolute temperature when analyzed with stochastic computer simulations. However, when the data were analyzed with isotropic Lorentzian line shape approximations, nonlinear plots were obtained.     

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.     

Identification of metal-binding proteins in human hepatoma lines by immobilized metal affinity chromatography and mass spectrometry

The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used matrix-assisted laser desorption ionization and electrospray ionization quadrupole time-of-flight mass spectrometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding proteins or peptides on an immobilized metal affinity chromatography column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding proteins identified included enolase, albumin, transferrin, and alcohol dehydrogenase as well as certain intracellular chaperone proteins. The Cu metalloproteome was not identical to the Zn metalloproteome. Peptide binding experiments demonstrated that Cu coordination prefers the order of residues histidine > methionine > cysteine. Although the Cu metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphorylethanolamine was identified as a post-translational modification at residue Glu-301 of elongation factor 1-alpha in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphosphoryl side group. These findings suggest that post-translational modification could be affected by intracellular actions of copper. Comparison of the Cu and Zn metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap (Seow, T. K., et al. (2001) Proteomics 1, 1249-1263). Proteins in the metalloproteomes of human hepatocytes can be identified by these methods. Variations in these metalloproteomes may have important physiological relevance.     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

DNA-binding proteins from Novikoff hepatoma cells

In 0.05 M NaCl, 6–8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5–1% of the total protein exhibits a 7–17-fold preference for rat DNA over Escherichia coli DNA. 1–1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with ¹²⁵I or ¹³¹I and characterized by sodium dodecylsulfate—polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Identification of phosphatidylserine-binding proteins in human white blood cells.

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Distribution of oxidized and HNE-modified proteins in U87 cells

Protein modification is one of the important processes during oxidative stress. This modification of proteins is either due to direct oxidation of proteins by various oxidants or due to secondary modification by lipid peroxidation products, e.g. 4-hydroxynonenal. In the here presented work we compare the intracellular distribution of protein modification products after treatment of human U87 astrocytoma cells with hydrogen peroxide or HNE. The treatment with hydrogen peroxide leads mainly to a cytosolic formation of oxidized proteins whereas HNE treatment is forming HNE-adducts throughout the cell. Therefore, we concluded that HNE diffusion distance in cells enables this lipid peroxidation product to act as a second messenger within the cell and on the other hand is the reason for the genotoxic properties of this compound.