| 1. |
- Neve, Etienne P A, et al.
(author)
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Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria
- 2012
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:9, s. 6307-6317
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Journal article (peer-reviewed)abstract
- Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase).
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| 2. |
- Neve, Etienne P. A., et al.
(author)
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An Integrated in Vitro Model for Simultaneous Assessment of Drug Uptake, Metabolism, and Efflux
- 2013
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 10:8, s. 3152-3163
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Journal article (peer-reviewed)abstract
- The absorption, distribution, metabolism, and excretion (ADME) of drugs in vivo are to a large extent dependent on different transport and metabolism routes. Elucidation of this complex transport-metabolism interplay is a major challenge in drug development and at present no in vitro models suitable for this purpose are at hand. The aim of this study was to develop flexible, well-controlled, easy-to-use, integrated cell models, where drug transport and drug metabolism processes could be studied simultaneously. HEK293 cells stably transfected with the organic anion transporting polypeptide 1B1 (OATP1B1) were subjected to either transient transfection or adenoviral infection to introduce the genes expressing cytochrome P450 3A4 (CYP3A4), NADPH cytochrome P450 oxidoreductase (POR), cytochrome b(5) (CYB5A), and multidrug resistance protein 1 (MDR1), in different combinations. Thereafter, the time and concentration dependent transport and metabolism of two well-characterized statins, atorvastatin (acid and lactone forms) and simvastatin (acid form), were determined in the different models. The results show that CYP3A4-dependent metabolism of the more hydrophilic atorvastatin acid was dependent on OATP1B1 uptake and influenced by MDR1 efflux. In contrast, the metabolism of the more lipophilic atorvastatin lactone was not affected by active transport, whereas the metabolism of simvastatin acid was less influenced by active transport than atorvastatin acid. Our results, together with the models being applicative for any combination of drug transporters and CYP metabolizing enzymes of choice, provide proof-of-concept for the potential of the new integrated cell models presented as valuable screening tools in drug discovery and development
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| 3. |
- Neve, Etienne P A
(author)
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Molecular basis and structural determinants for the cellular localization of cytochrome P450 2E1
- 2000
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Doctoral thesis (other academic/artistic)abstract
- Cytochrome P450 2E1 (CYP2E1) is the alcohol inducible member of the P450 family and has a role in gluconeogenesis under fasting conditions. However, CYP2E1 is mainly of toxicological significance and metabolizes xenobiotics to their carcinogenic and toxic metabolites and generates active oxygen species that can cause lipid peroxidation and apoptosis, processes observed in alcoholic liver disease (ALD). CYP2E1 like other xenobiotic metabolizing P450s, is mainly located in the membrane of the endoplasmic reticulum (ER), where it is anchored and retained through its hydrophobic NH2-terminus, leaving the large COOH-terminal domain including the catalytic site exposed to the cytosol. CYP2E1 has also been detected in other cellular compartments such as lysosomes and the plasma membrane. CYP2E1 located at the plasma membrane has been suggested to play a role in the immune mediated hepatotoxicity observed in patients suffering from ALD. Other forms of P450 have also been observed at the extracellular side of the plasma membrane and this could be important in the etiology of several forms of drug-induced hepatitis. During the past years much controversy existed regarding the topology of P450s on the plasma membrane and the molecular mechanism behind the appearance of P450 at the cell surface has remained obscure. The involvement of the Golgi apparatus has been implied in the transport of P450s to the plasma membrane in isolated rat hepatocytes, although reports exist where investigators were unable to detect P450 in the Golgi apparatus. Therefore we isolated the Golgi apparatus from rat liver and the distribution of several P450 enzymes in three subfractions of the Golgi apparatus was studied. It was found that there was an enzyme specific appearance of P450 in the isolated Golgi fractions: CYP2E1 CYP4A1 and CYP1A2 together with NADPH cytochrome P450 reductase were present, whereas CYP3A1 was not. This suggested that CYP3A1 was efficiently retained in the ER membrane and others, like CYP2E1, were transported out of the ER to the Golgi apparatus. The molecular mechanism responsible for the exit from the ER and subsequent transport to the plasma membrane of CYP2E1 was studied by constructing several NH2-terminal mutants, since the NH2-terminal domain has been demonstrated to be important for targeting to and retention in the ER membrane. The presence of CYP2E1 and its mutants at the outer surface of the plasma membrane was established by several techniques such as immunofluorescent microscopy and cell surface biotinylation studies. It was demonstrated that N++2E1, which carried two positively charged residues in the NH2-terminus, was present at the plasma membrane at twice the amount as wild-type CYP2E1 Protease protection experiments further demonstrated that CYP2E1 was incorporated in two topologies in the ER membrane, most of the protein was in the expected cytoplasmic orientation while a small fraction (about 2%) was incorporated in a lumenal orientation. N++2E1 appeared to have a higher extent of lumenal orientation than the other variants. Removal or modification of the hydrophobic NH2-terminal transmembrane domain of CYP2E1 resulted in specific targeting to the mitochondria. After mitochondrial import and processing, a soluble and catalytically active protein, called [delta]2E1 (Mr ¡Ö 40 kD), was formed. Low levels of [delta]2E1 were also observed in mitochondria isolated from rat liver, thus showing that [delta]2E1 is present in vivo. Removal or modification of the NH2-terminus of CYP2E1 results in the exposure of a mitochondrial targeting signal that directs the protein to the mitochondria. The mitochondrial targeting signal was identified and demonstrated to be located between amina acid residues 74 and 95, an area rich in positively charged amino acid residues and also containing a hydrophobic region. The role of the NH2-terminus of CYP2E1 in the intracellular targeting was investigated using Saccharomyces cerevisiae as a model system. Wild-type CYP2E1 was shown to be expressed in the ER, while modification of the NH2-terminal transmembrane domain did not result in mitochondrial targeting, as was observed in the mammalian cell line. Furthermore, removal of the NH2-terminal transmembrane domain resulted in mitochondrial targeting but not in processing to the mature form [delta]2E1. The same region that was identified to be responsible for mitochondrial targeting in mammalian cells was also demonstrated to be responsible for directing these NH2-terminally truncated forms of CYP2E1 to the mitochondria in yeast. In addition to the NH2-terminal part, the region between as 64 and 95 was shown to cause membrane interactions of the protein. It is suggested that yeast can be a useful model system as a complement to mammalian cells for studying the intracellular protein targeting of mammalian proteins, but differences such as in the mitochondrial import and processing, do exist. It is concluded that the hydrophobic NH2-terminal transmembrane domain of CYP2E1 plays an important role in the cellular targeting. Removal or modification of this region resulted in the targeting of the protein to the mitochondria where, after import, it was processed to a soluble and catalytically active form. The presence of CYP2E1 at the extracellular side of the plasma membrane was unequivocally demonstrated and the molecular mechanism behind its appearance was resolved by showing the lumenal incorporation of a small fraction of the enzyme during its synthesis, that is not efficiently retained in the ER and transported via the Golgi apparatus to the plasma membrane.
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| 4. |
- Shimoji, Miyuki, et al.
(author)
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Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1
- 2017
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In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1859:2, s. 238-244
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Journal article (peer-reviewed)abstract
- Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.
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| 5. |
- Vincent, Theresa, et al.
(author)
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A SNAIL1–SMAD3/4 transcriptional repressor complex promotes TGF‑β mediated epithelial–mesenchymal transition
- 2009
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In: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 11:8, s. 943-950
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Journal article (peer-reviewed)abstract
- Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.
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| 6. |
- Överby, Anna K, et al.
(author)
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Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae) : a useful system for studying bunyaviral packaging and budding
- 2006
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In: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 80:21, s. 10428-10435
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Journal article (peer-reviewed)abstract
- In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.
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| 7. |
- Överby, Anna K, et al.
(author)
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The cytoplasmic tails of Uukuniemi Virus (Bunyaviridae) G(N) and G(C) glycoproteins are important for intracellular targeting and the budding of virus-like particles
- 2007
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In: Journal of Virology. - 0022-538X .- 1098-5514. ; 81:20, s. 11381-11391
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Journal article (peer-reviewed)abstract
- Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V. Popov, E. P. Neve, and R. F. Pettersson, J. Virol. 80:10428-10435, 2006). We previously reported that information necessary for the packaging of ribonucleoproteins into VLPs is located within the G(N) cytoplasmic tail (A. K. Overby, R. F. Pettersson, and E. P. Neve, J. Virol. 81:3198-3205, 2007). The G(N) glycoprotein cytoplasmic tail specifically interacts with the ribonucleoproteins and is critical for genome packaging. In addition, two other regions in the G(N) cytoplasmic tail, encompassing residues 21 to 25 and 46 to 50, were shown to be important for particle generation and release. By the introduction of point mutations within these two regions, we demonstrate that leucines at positions 23 and 24 are crucial for the initiation of VLP budding, while leucine 46, glutamate 47, and leucine 50 are important for efficient exit from the endoplasmic reticulum and subsequent transport to the Golgi complex. We found that budding and particle generation are highly dependent on the intracellular localization of both glycoproteins. The short cytoplasmic tail of UUK G(C) contains a lysine at position -3 from the C terminus that is highly conserved among members of the Phlebovirus, Hantavirus, and Orthobunyavirus genera. Mutating this single amino acid residue in G(C) resulted in the mislocalization of not only G(C) but also G(N) to the plasma membrane, and VLP generation was compromised in cells expressing this mutant. Together, these results demonstrate that the cytoplasmic tails of both G(N) and G(C) contain specific information necessary for efficient virus particle generation.
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| 8. |
- Överby, Anna K, et al.
(author)
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The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging
- 2007
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In: Journal of Virology. - 0022-538X .- 1098-5514. ; 81:7, s. 3198-3205
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Journal article (peer-reviewed)abstract
- We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G(N) cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.
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