Human inter-alpha-trypsin inhibitor. Synthesis and maturation in hepatoma HepG2 cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Bourguignon J
PROVIDER: S-EPMC1138820 | biostudies-other | 1989 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.
| S-EPMC1137266 | biostudies-other
Project description:Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of (18)O water. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.
| S-EPMC4084840 | biostudies-literature
Project description:Retinoic acid-related Orphan Receptor alpha (ROR?; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by ROR? in liver cells by generating HepG2 human hepatoma cells stably over-expressing ROR?. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which ROR? was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by ROR?, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by ROR?. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by ROR?. SPARC was found to be a new putative ROR? target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by ROR? also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by ROR? in HepG2 cells. Therefore these genes must now be considered as direct ROR? targets. Our results open new routes on the roles of ROR? in glucose metabolism and carcinogenesis within cells of hepatic origin.
| S-EPMC3144224 | biostudies-literature
Project description:The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-alpha-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury.To determine whether IaI promotes HA-mediated angiogenesis in tissue injury.An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined.IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC.Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis. Clinical trial registered with www.clinicaltrials.gov (NCT00016627).
| S-EPMC2577729 | biostudies-literature
Project description:?-Tocopherol transfer protein (?-TTP) is a ~32 kDa protein expressed mainly in hepatocytes. The major function of the protein is to bind specifically to ?-tocopherol and, together, the complex transfers from late lysosomes to the cell membrane. A previous study indicated that some factors might be required in the transferring process. However, there is little information available about the potential transferring factors. In addition, there remains much to learn about other physiological processes which ?-TTP might participate in. Thus, in this study a human ?-TTP eukaryotic expression vector was successfully constructed and expressed in human hepatoma cells (HepG2). The sensitive genes related to ?-TTP were then screened by microarray technology. Results showed that expression of the vector in HepG2 cells led to the identification of 323 genes showing differential expression. The differentially expressed transcripts were divided into four main categories, including (1) cell inflammation; (2) cell cycle and cell apoptosis; (3) cell signaling and gene regulation; and (4) cellular movement. A few cellular movement related transcripts were selected and verified by quantitative real-time PCR. Expressions of some were significantly increased in ?-TTP-expressed group, which indicated that these factors were likely to play a role in the transferring process.
| S-EPMC4964392 | biostudies-literature
Project description:Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.
| S-EPMC1135053 | biostudies-other
Project description:Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C1-chrysenes are representative PAHs present in the crude oil and have been detected in contaminated sea food in amounts that exceed their permissible safety thresholds. We describe the metabolism of the most carcinogenic C1-chrysene regioisomer, 5-methylchrysene (5-MC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 5-MC-tetraol, a signature metabolite of the diol-epoxide pathway, was identified as reported previously. Novel O-monosulfonated-5-MC-catechol isomers and O-monomethyl-O-monosulfonated-5-MC-catechol were discovered, and evidence for their precursor ortho-quinones was obtained. The identities of O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione were validated by comparison to authentic synthesized standards. Dual metabolic activation of 5-MC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones is reported for the first time. Evidence was also obtained for minor metabolic conversion of 5-MC to form monohydroxylated-quinones and bis-phenols. The identification of 5-MC-tetraol, O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione supports metabolic activation of 5-MC by P450 and AKR isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by COMT and SULT isozymes. The major metabolites, O-monosulfonated-catechols and tetraols, could be used as biomarkers of human exposure to 5-MC resulting from oil spills.
| S-EPMC4709222 | biostudies-literature
Project description:Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor ?1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function.
| S-EPMC2869104 | biostudies-literature
Project description:In the past few decades, the incidence of liver cancer has been rapidly rising across the world. Rosemary is known to possess antioxidant activity and is used as natural antioxidant food preservative. It is proposed to have anticancer activity in treating different tumor models. In this study, we try to explore the impact of rosemary extracts on upregulating the level of Nrf2 and Nrf2-regulatory proteins, Sestrin2 and MRP2 in HepG2 cells, and to speculate its potential mechanism. The anticancer activity of rosemary extract, including its polyphenolic diterpenes carnosic acid and carnosol, was evaluated to understand the potential effect on HepG2 cells. Rosemary extract, carnosic acid, and carnosol induced the expression of Sestrin2 and MRP2 associate with enhancement of Nrf2 protein level in HepG2 cells, in which carnosic acid showed most obvious effect. Although the activation pathway of Nrf2/ARE was not exactly assessed, it can be assumed that the enhancement of expression of Sestrin2 and MRP2 may result from upregulation of Nrf2.
| S-EPMC5327778 | biostudies-literature
Project description:As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided.
| S-EPMC5192242 | biostudies-literature