scholarly journals Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself

1993 ◽  
Vol 293 (2) ◽  
pp. 437-442 ◽  
Author(s):  
L Mach ◽  
H Schwihla ◽  
K Stüwe ◽  
A D Rowan ◽  
J S Mort ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Mammalian Cells ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Human Hepatoma ◽  
Single Chain ◽  
Human Hepatoma Cells ◽  
Chain Form

In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.

scholarly journals Binding of FAD to 6-hydroxy-d-nicotine oxidase apoenzyme prevents degradation of the holoenzyme

1989 ◽  
Vol 258 (1) ◽  
pp. 187-192 ◽  
Author(s):  
R Brandsch ◽  
V Bichler ◽  
B Krauss
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cysteine Proteinase Inhibitor ◽  
Western Blots ◽  
E Coli ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Cloned Gene

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

scholarly journals Loss of Heterochromatin Protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death

2002 ◽  
Vol 115 (9) ◽  
pp. 1803-1813 ◽  
Author(s):  
Ilaria Filesi ◽  
Alessio Cardinale ◽  
Sjaak van der Sar ◽  
Ian G. Cowell ◽  
Prim B. Singh ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Wide Spectrum ◽  
Human Fibroblasts ◽  
Protein S ◽  
Single Chain ◽  
Heterochromatin Protein 1 ◽  
Apoptotic Pathway

The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.

scholarly journals Evaluation of an In vitro Approach to the Prediction of In vivo Effects on Multidrug Resistance in Human Hepatoma Cells

2018 ◽  
Vol 09 (02) ◽  
Author(s):  
Kosuke Inamura ◽  
Kazumi Emoto ◽  
Hideaki Ichihara ◽  
Kohei Sasaki ◽  
Takuya Iwasa ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Hepatoma Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
In Vivo Effects

Non-genomic action of TCDD to induce inflammatory responses in HepG2 human hepatoma cells and in liver of C57BL/6J mice

2010 ◽  
Vol 391 (10) ◽  
Author(s):  
Wen Li ◽  
Christoph F.A. Vogel ◽  
Dalei Wu ◽  
Fumio Matsumura
Keyword(s):  
Hepg2 Cells ◽  
Inflammatory Responses ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Classical Action ◽  
Cox 2

Abstract To assess the significance of the non-genomic signaling of TCDD (=dioxin) on liver of C57BL/6 mice and HepG2 human hepatoma cells, we first determined the group of markers that are susceptible to inhibition by parthenolide, a compound known to specifically suppress NF-κB-mediated inflammation. Of those, the most consistent marker turned out to be SOCS3 (a suppressor of cytokine signaling) known to respond to inflammation. An early diagnostic test on the action of TCDD on HepG2 cells in vitro within 3–6 h indicated that Cox-2 and SOCS3 are mainly induced via a non-genomic route, whereas PAI-2 appears to be induced through the classical action route. More detailed diagnostic tests at later stages of action of TCDD in HepG2 cells revealed that induction of IL-1β, BAFF, and iNOS are largely mediated by the protein kinase-dependent non-genomic route. An in vivo study on the 7 day action of TCDD on liver of AhRNLS mice showed that several early markers (e.g., Cox-2, MCP-1 and SOCS3) are induced, but not late markers such as IL-1β. Together, these results show that the non-genomic pathway contributes significantly to the early stress response reactions to TCDD that includes inflammation in hepatoma cells as well as in the liver.

Anti-proliferative activity of fenretinide in human hepatoma cells in vitro and in vivo

2007 ◽  
Vol 18 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Jianzhong Shentu ◽  
Bo Zhang ◽  
Lingling Fan ◽  
Qioajun He ◽  
Bo Yang ◽  
...  
Keyword(s):  
Proliferative Activity ◽  
Hepatoma Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells

Effect of Cysteine Proteinase Inhibitors on Murine B16 Melanoma Cell Invasion in vitro

2002 ◽  
Vol 383 (5) ◽  
pp. 839-842 ◽  
Author(s):  
Natasa Sever ◽  
Metka Filipic ◽  
Joze Brzin ◽  
Tamara T. Lah
Keyword(s):  
Cell Invasion ◽  
Cathepsin B ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Cysteine Proteinase Inhibitors

Abstract Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

scholarly journals Proteolytic processing and glycosylation of cathepsin B. The role of the primary structure of the latent precursor and of the carbohydrate moiety for cell-type-specific molecular forms of the enzyme

1992 ◽  
Vol 282 (2) ◽  
pp. 577-582 ◽  
Author(s):  
L Mach ◽  
K Stüwe ◽  
A Hagen ◽  
C Ballaun ◽  
J Glössl
Keyword(s):  
Heavy Chain ◽  
Cathepsin B ◽  
Hepg2 Cells ◽  
Cysteine Proteinase ◽  
Carbohydrate Moiety ◽  
Molecular Forms ◽  
Cell Type ◽  
Single Chain ◽  
Cell Type Specific ◽  
Chain Form

The lysosomal cysteine proteinase cathepsin B is synthesized in cultured human hepatoma HepG2 cells as an inactive 44 kDa precursor and subsequently processed to the mature single-chain enzyme with a molecular mass of 33 kDa. Intralysosomal conversion into the two-chain form results in subunits of 27 kDa, 24 kDa (heavy chain) and 5 kDa (light chain). Enzymic deglycosylation reveals that the 27 kDa polypeptide is the glycosylated variant of the carbohydrate-free 24 kDa heavy-chain form. The intracellular transport to the lysosomes is dependent upon mannose 6-phosphate-containing N-linked oligosaccharides. Receptor-mediated endocytosis of human skin-fibroblast-derived procathepsin B by HepG2 cells resulted in processed molecular forms that are not distinguishable from endogenous cathepsin B, thus favouring rather a cell-type-specific processing than structural differences due to the source of the proenzyme. The conversion step of single-chain catehpsin B into the two-chain enzyme is inhibited in vivo by the irreversible cysteine-proteinase inhibitors Z-Phe-Ala-CHN2 and, albeit weaker, Z-Phe-Phe-CHN2. Both substances have no effect on the activation of procathepsin B to the mature enzyme. The carbohydrate moiety of cathepsin B exerts no significant influence on the stability and the enzymatic activity of the enzyme.

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