Human Liver Proteins Research Articles

Ligand specificity and conformational stability of human fatty acid-binding proteins

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.

Distribution of some rare earth elements and their binding species with proteins in human liver studied by instrumental neutron activation analysis combined with biochemical techniques

Distribution patterns of three rare earth elements and their binding species with proteins in the human liver samples were studied by instrumental neutron activation analysis combined with biochemical techniques. It was indicated that La, Ce and Sm were highly concentrated in microsomes, although the total content of this fraction was the least among all of the liver organelles, which was similar with the behavior of Ca and Fe. More than 85% of La, Ce and Sm was present in the cytosolic protein fractions separated by ammonium sulfate and ethanol. It indicated that these elements were mainly incorporated into protein. It was also revealed by means of size-exclusion chromatography that at least three La-binding proteins ( M w: 335±50, 94.5±15.4, 13.6±3.8 kD), three Ce-binding proteins ( M w: 335±50, 85.1±12.0, 22.8±6.3 kD) and about four Sm-binding proteins ( M w: 335±70, 82.1±5.4, 32.3±5.8 and 13.6±4.5 kD) were present in the supernatant fraction of human liver. Most of La, Ce and Sm were found in the high-molecular-weight protein region.

Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver

Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver

Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver

Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver

NRH:quinone oxidoreductase2 (NQO2)

The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25 956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.

In Vitro Covalent Binding of Nafenopin–CoA to Human Liver Proteins

Endogenous fatty acyl–CoAs play an important role in the acylation of proteins. A number of xenobiotic carboxylic acids are able to mimic fatty acids, forming CoA conjugates and acting as substrates in pathways of lipid metabolism. In this study nafenopin, a substrate for human hepatic fatty acid–CoA ligases, was chosen as a model compound to study xenobiotic acylation of human liver proteins. 3H-nafenopin (± unlabeled palmitate) or 14C-palmitate (± unlabeled nafenopin) were incubated for up to 120 min at 37°C with ATP, CoA, and homogenate protein (1 mg/ml) from four individual human livers. Nafenopin covalently bound to proteins was detectable in all human livers and increased with time. Nafenopin adduct formation was directly proportional to nafenopin–CoA formation (r = 0.985, p < 0.05). Attachment of nafenopin to proteins involved both thioester and amide linkages with 76 and 24% of adducts formed with proteins > 100 and 50–100 kDa, respectively. Protein acylation by palmitate was also demonstrated. Palmitate significantly inhibited nafenopin–CoA formation by 29% but had no effect on nafenopin–CoA-mediated protein acylation. In contrast, nafenopin significantly inhibited protein palmitoylation by palmitoyl–CoA. This is the first study to demonstrate a direct relationship between xenobiotic–CoA formation, acylation of human liver proteins, and inhibition of endogenous palmitoylation. The ability of xenobiotics to acylate tissue proteins may have important biological consequences including perturbation of endogenous regulation of protein localization and function.

Localization of the Wilson's disease protein in human liver

Background & Aims: Wilson's disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilson's disease protein in hepatic copper handling, its localization in human liver was investigated. Methods: By use of a specific antibody, localization of the Wilson's disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. Results: The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. Conclusions: These results show that in human liver the Wilson's disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion. GASTROENTEROLOGY 1999;117:1380-1385

Exon-intron organization of the human multidrug-resistance protein 2 ( MRP2) gene mutated in Dubin–Johnson syndrome

Background & Aims: The Dubin–Johnson syndrome is characterized by conjugated hyperbilirubinemia and by impaired secretion of anionic conjugates from hepatocytes into bile. Absence of the multidrug-resistance protein 2 (MRP2; symbol ABCC2), an adenosine triphosphate–dependent conjugate export pump, from the hepatocyte canalicular membrane is the molecular basis of this syndrome. The aim of this study was the elucidation of all exon-intron boundaries of the MRP2 gene as a prerequisite for the analysis of mutations in patients with Dubin–Johnson syndrome. Methods: Exon-intron boundaries of MRP2 were determined, and the amplified exons were screened for mutations. Immunofluorescence microscopy served to localize the MRP2 protein in human liver. Results: The human MRP2 gene is ~45 kilobases long; it contains 32 exons and a high proportion of class 0 introns. In 2 patients with Dubin–Johnson syndrome, we detected a nonsense mutation at codon 1066 and a 6-nucleotide deletion mutation affecting codons 1392–1394. The MRP2 protein was absent from the canalicular membrane of both patients. Conclusions: The mutations detected so far show that various mutations in the MRP2 gene can lead to the Dubin–Johnson syndrome. The exon-intron boundaries established in this article will facilitate the analysis of additional mutations in the MRP2 gene. GASTROENTEROLOGY 1999;117:653-660

The growth hormone receptor associates with Jak1, Jak2 and Tyk2 in human liver.

Studies in cell lines have shown that Jak2 is the primary tyrosine kinase involved in signal transduction by the growth hormone receptor (GHR). In addition, growth hormone (GH) stimulates tyrosine phosphorylation of Jak1 and Jak3 in certain cell lines, while the effect on Tyk2 has not been analysed. We have investigated the expression of Jak proteins in human liver and analysed their interactions with the GHR. Using Western blot analysis and immunohistochemistry, we demonstrate that Jak1, Jak2, Jak3 and Tyk2 are present in human liver. Immunoprecipitation by an antibody against the GHR (Mab 263) followed by immunoblotting with specific antibodies against Jak proteins showed that Jak1, Jak2 and Tyk2 were associated with the GHR in this tissue. We conclude that the GHR associates with Jak1, Jak2 and Tyk2 in human liver. Although experiments in vitro indicate that Jak2 mediates GH signalling, our results open the possibility that other Jak proteins may influence GHR signalling in human liver.

Subcellular distribution of selenium and Se-containing proteins in human liver

Selenium is an essential trace element in many living organisms. In the present paper, the subcellular distribution of selenium and Se-containing proteins in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation and column chromatography. Selenium was mainly enriched in nuclei, mitochondria and cytosol. Almost half of Se existed in the nuclei due to their large amount in liver and high Se concentration. 15–30% of Se was found in small compounds with M r<2000 in the liver components separated by dialysis. The average abundance of Se in small molecular mass species of whole-liver was 23.6%, which suggested most of Se associated with biological macromolecules. Eight kinds of Se-containing proteins with molecular mass of 335±20, 249±15, 106±11, 84.6±5.8, 70.5±5.4, 45.6±1.5, 14.8±2.6, 8.5±1.2 kDa were found in the subcellular fractions of human liver. Among them the 335, 84.6 and 8.5 kDa proteins were individually present in one subcellular fraction, whereas the others coexisted in two, three or four subcellular fractions. The most abundant Se-containing proteins, 70.5 and 14.8 kDa, accounted for 33.6% and 48.5% in the whole-liver soluble Se-containing protein, respectively. The former was enriched in cytosol and the latter was mainly present in nuclei and mitochondria.

Importance of metabolism in pharmacological studies: possible in vitro predictability

Metabolic transformation of drug leads to the formation of a large number of secondary compounds. These metabolites may (a) participate to the elimination of the patent drug, (b) have similar or different therapeutic effects compared to the parent drug (c) exert toxic effects. Cytochromes P450 are the main enzymes involved in the biotransformation of exogenous drugs, leading to oxidized, reduced or peroxidized metabolites. Different isozymes of P450 are present in already all the organs and differ by their affinity for substrate families. P450 3A is the most abundant P450 protein in the adult human liver and is able to transform hundreds of substrates into either drugs or endogenous compounds such as testosterone. Its catalytic activities are regulated either by induction or by inhibition. Attempts to predict metabolic transformation of a given drug are based on the amount of P450 expressed in heterologous systems, induction, and inhibition experiments and by comparison to classical P450 substrates. Erythromycin metabolism and its P450 effects are used to illustrate the complexity and the consequences of metabolic transformation of a given drug.

Serum IgA antibodies to human gut luminal aspirates and human liver in alcoholic liver disease.

Elevation of serum IgA is a characteristic feature of alcoholic liver disease. It has been proposed that this occurs partly as an antigenic response to gut-derived proteins or acetaldehyde-modified liver proteins, but the principal antigens responsible remain unknown. The goal of this study was to determine if serum IgA antibodies were present against human gut luminal antigens or liver antigens in alcoholic liver disease. Twenty-nine patients with alcoholic liver disease, 10 with primary biliary cirrhosis, 12 with "other" liver diseases, 8 alcoholics, and 20 healthy subjects were studied. Western blotting was used to examine the reactivity of sera from these groups against human small and large bowel aspirates and liver tissue from alcoholic liver disease patients. Serum IgA antibodies to a 140 kDa colonic luminal protein were found in 22 (76%) patients in the alcoholic liver disease group (p < 0.0001), and 7 (24%) patients had serum IgA antibodies to a 40 kDa colonic luminal protein (p = 0.04). These responses were confined to colonic aspirates and not observed in other disease groups, alcoholics or healthy subjects. There was no significant serum IgA response to human liver proteins in alcoholic liver disease. Serum IgA antibodies to a human 140 kDa colonic luminal protein are frequently found in alcoholic liver disease. This novel antigen may contribute to the increased levels of circulating IgA in alcoholic liver disease.

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture.

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1-12-3 recognized only one protein of identical mobility on the two-dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.

Dehydroepiandrosterone sulfate (DHEAS): identification of a carrier protein in human liver and brain

Dehydroepiandrosterone sulfate (DHEAS) is the major circulating steroid in man. Pharmacologically, it exerts marked neuropsychiatric effects. Since no target receptor has been identified, we investigated whether the organic anion transporting polypeptide (OATP), a multispecific steroid carrier, transports DHEAS. Expression of the human liver OATP in Xenopus laevis oocytes resulted in high-affinity, partially Na +-dependent uptake of [ 3H]DHEAS ( K m: 6.6 μmol/l). DHEAS transport was inhibited by bromosulfophthalein, bile acids, sulfated estrogens and dexamethasone. Northern blot analysis showed widespread expression of OATP in human brain. These data identify OATP as the first known target protein of DHEAS in human liver and brain.

Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

P.43 The effects of short term parenteral nutrition on human liver protein metabolism

P.43 The effects of short term parenteral nutrition on human liver protein metabolism

Distribution of lactoferrin and 60/65 kDa heat shock protein in normal and inflamed human intestine and liver.

Immunisation against the mycobacterial heat shock protein (hsp-65) has been proposed to lead to production of autoantibodies against human lactoferrin. Such antibodies occur in ulcerative colitis and in primary sclerosing cholangitis. This study analysed the distribution of hsp-65 and lactoferrin in biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis and studied whether immunisation against mycobacterial hsp-65 resulted in production of antilactoferrin antibodies and vice versa. Polyclonal rabbit antihuman lactoferrin and monoclonal mouse anti-hsp-65 (ML30) were used for immunohistochemistry on biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis. Rats were immunised against human lactoferrin and mycobacterial hsp-65 respectively. Antibody measurements were done by enzyme immunosorbent assays. It was found that lactoferrin and hsp-60/65 were not codistributed. Lactoferrin was found on vascular endothelium and in nonparenchymal liver cells both in inflamed and uninflamed tissues, but only in the hepatocytes of inflamed liver. ML30 reactivity was not inhibited by antilactoferrin antibodies. Rat anti-hsp-65 serum had no detectable antilactoferrin antibodies. In conclusion, antilactoferrin antibodies probably do not arise by immunisation against mycobacterial hsp-65. Both nonparenchymal cells and hepatocytes probably participate in clearance of lactoferrin. Endothelial exposure of lactoferrin may have pathogenic implications in diseases with antilactoferrin autoantibodies.

Increased S-100 protein-immunoreactivity of Kupffer cells is associated with lymphohematological malignancy.

The distribution of S-100 protein in normal tissue has been studied extensively. However, little is known about its expression in pathologic states. The aim of the present study was to investigate the expression of S-100 protein in diseased human liver, especially in Kupffer cells. One hundred cases of autopsy livers originating from patients with various diseases were examined. Increased S-100-immunoreactivity of Kupffer cells was observed in six cases. Of the six cases, four were derived from a lymphohematologic malignancy, such as B cell lymphoma, B cell lymphoblastic leukemia, multiple myeloma and chronic myelogenous leukemia with lymphoblastic crisis. Lymphohematologic malignancy accounted for 16 out of the 100 cases examined. Thus, increased S-100-positive Kupffer cells was significantly associated with lymphohematologic malignancy (P < 0.01); 25% (4/16) in cases with lymphohematologic malignancy versus 2.4% (2/84) in the remaining cases. Moreover, some of these S-100-positive Kupffer cells were positive for S-100 beta-subunit, which is not normally expressed by Kupffer cells. Although the reason for this increased S-100-immunoreactivity is speculative, the authors' hypothesis is that tumor cells may produce some factor(s) that induce the expression of S-100 protein in Kupffer cells.

Age but not gender selectively affects expression of individual cytochrome P450 proteins in human liver

Multivariate linear regression analysis was used to examine the influence of age, gender and environmental variables on the hepatic content of cytochromes P450 (CYP, P450) 1A2, 2C, 2E1 and 3A in 71 subjects; 21 with histologically normal livers and 50 with chronic liver disease. There was a clear negative association between age and total P450 content, NADPH-cytochrome c reductase activity and levels of 2E1 and 3A proteins. 1A2 and 2C proteins were unaltered with advancing age. Gender did not influence the expression of any of the CYP proteins. Cigarette smoking was associated with enhanced levels of 1A2, but effects of drug ingestion and alcohol consumption were not apparent in this study, probably because of case selection. It is concluded that age but not gender is a constitutional factor that influences the hepatic content of cytochrome P450 and selected CYP proteins.