Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

Caractérisation de la propriété de la protéine ZEBRA du virus Epstein-Barr à pénétrer dans les cellules

The basic-leucine zipper (bZIP) transcriptional activator ZEBRA of the Epstein - Barr virus was recently shown to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. During this PhD study, I investigated the potential application of ZEBRA as a transporter protein to facilitate transduction of cargo proteins. The analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization was spanning residues 178-220. The MD efficiently transported reporter proteins, such as EGFP and β-galactosidase, into several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by β-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in presence of heparin. Furthermore, internalization was blocked at 4 °C, whereas no ATP was required as witnessed by an only 25 % decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors had no significant impact on MD-EGFP uptake. Only methyl-β-cyclodextrin (MβCD) inhibited MD-EGFP uptake by 40 % indicating the implication of the lipid raft mediated endocytotic pathway. These data suggest that ZEBRA-MD-reporter protein transduction occurs mostly via direct translocation through the cell membrane and not by endocytosis. Tissue distribution of ZEBRA-MD-EGFP or ZEBRA-MD-β-galactosidase was analyzed after administration into mice. ZEBRA coupled reporter proteins were found in single cells of various tissues contrary to application of EGFP and β-galactosidase alone. In addition, the cell penetrating sequence of ZEBRA (MD) was fused to anti-tumor protein IL-24/MDA-7. Induced cell death after successful internalization of MD-IL-24/MDA-7 was proven by the cleavage of apoptosis specific caspases without showing difference between normal and tumor breast cells. The mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins.Il a été récemment démontré que l'activateur de transcription ZEBRA du virus Epstein-Barr contenant un motif "basic-leucine zipper (bZIP)" traverse la membrane externe des cellules vivantes et s'accumule dans le noyau des lymphocytes. Durant mon travail de thèse, j'ai étudié la possibilité d'utiliser ZEBRA comme protéine de transport afin de faciliter la transduction de protéines cargo. L'analyse de différentes formes tronquées de ZEBRA a permis de mettre en évidence que le domaine minimal (MD) nécessaire à l'internalisation inclut les résidus 178-220. Le MD a permis de transporter de manière efficace des protéines rapporteur comme la EGFP et la -galactosidase dans plusieurs lignées cellulaires normales et tumorales. La fonction des protéines cargo internalisées a été confirmée par l'activité -galactosidase dans les cellules transduites, et aucune toxicité cellulaire associée au MD n'a été détectée. La translocation du MD à travers la membrane cellulaire nécessite la liaison aux héparanes sulfates protéoglycans associés à la surface de la cellule comme cela a été démontré par la forte inhibition du transport de protéines en présence d'héparine. En outre, l'internalisation est également bloquée à basse température (4 °C). De plus, une réduction de seulement 25 % du transport de protéines cargo dans des cellules avec des stocks d'ATP épuisés démontre que l'internalisation est un processus indépendant de l'ATP. Les inhibiteurs classiques d'endocytose n'ont aucun effet significatif sur le transport de MD-EGFP. Seul le methyl--cyclodextrin inhibe de 40 % le transport de MD-EGFP, indiquant l'implication d'une voie d'endocytose médiée par les radeaux lipidiques. Ces résultats suggèrent que le transport de la protéine rapporteur ZEBRA-MD se produit principalement par translocation directe à travers la membrane cellulaire et non par endocytose. La distribution tissulaire d'EGFP ou de la β-galactosidase couplés ou non avec ZEBRA-MD a été étudiée après injection dans la souris. Seules les protéines de fusion avec ZEBRA-MD ont pu être révélées dans les cellules de différents tissus. De plus, la séquence de translocation de ZEBRA-MD a été fusionnée avec la protéine anti-tumorale IL-24/MDA-7. La mort cellulaire induite par l'internalisation de ZEBRA-MD-IL-24/MDA-7 a été mise en évidence par le clivage des caspases de la voie d'apoptose aussi dans des cellules normales que dans des cellules tumorales du sein. En conclusion, le mécanisme d'internalisation de ZEBRA-MD est approprié pour un transport efficace de protéines biologiquement actives

Caractérisation de la propriété de la protéine ZEBRA du virus Epstein-Barr à pénétrer dans les cellules

The basic-leucine zipper (bZIP) transcriptional activator ZEBRA of the Epstein - Barr virus was recently shown to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. During this PhD study, I investigated the potential application of ZEBRA as a transporter protein to facilitate transduction of cargo proteins. The analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization was spanning residues 178-220. The MD efficiently transported reporter proteins, such as EGFP and β-galactosidase, into several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by β-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in presence of heparin. Furthermore, internalization was blocked at 4 °C, whereas no ATP was required as witnessed by an only 25 % decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors had no significant impact on MD-EGFP uptake. Only methyl-β-cyclodextrin (MβCD) inhibited MD-EGFP uptake by 40 % indicating the implication of the lipid raft mediated endocytotic pathway. These data suggest that ZEBRA-MD-reporter protein transduction occurs mostly via direct translocation through the cell membrane and not by endocytosis. Tissue distribution of ZEBRA-MD-EGFP or ZEBRA-MD-β-galactosidase was analyzed after administration into mice. ZEBRA coupled reporter proteins were found in single cells of various tissues contrary to application of EGFP and β-galactosidase alone. In addition, the cell penetrating sequence of ZEBRA (MD) was fused to anti-tumor protein IL-24/MDA-7. Induced cell death after successful internalization of MD-IL-24/MDA-7 was proven by the cleavage of apoptosis specific caspases without showing difference between normal and tumor breast cells. The mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins.Il a été récemment démontré que l'activateur de transcription ZEBRA du virus Epstein-Barr contenant un motif "basic-leucine zipper (bZIP)" traverse la membrane externe des cellules vivantes et s'accumule dans le noyau des lymphocytes. Durant mon travail de thèse, j'ai étudié la possibilité d'utiliser ZEBRA comme protéine de transport afin de faciliter la transduction de protéines cargo. L'analyse de différentes formes tronquées de ZEBRA a permis de mettre en évidence que le domaine minimal (MD) nécessaire à l'internalisation inclut les résidus 178-220. Le MD a permis de transporter de manière efficace des protéines rapporteur comme la EGFP et la -galactosidase dans plusieurs lignées cellulaires normales et tumorales. La fonction des protéines cargo internalisées a été confirmée par l'activité -galactosidase dans les cellules transduites, et aucune toxicité cellulaire associée au MD n'a été détectée. La translocation du MD à travers la membrane cellulaire nécessite la liaison aux héparanes sulfates protéoglycans associés à la surface de la cellule comme cela a été démontré par la forte inhibition du transport de protéines en présence d'héparine. En outre, l'internalisation est également bloquée à basse température (4 °C). De plus, une réduction de seulement 25 % du transport de protéines cargo dans des cellules avec des stocks d'ATP épuisés démontre que l'internalisation est un processus indépendant de l'ATP. Les inhibiteurs classiques d'endocytose n'ont aucun effet significatif sur le transport de MD-EGFP. Seul le methyl--cyclodextrin inhibe de 40 % le transport de MD-EGFP, indiquant l'implication d'une voie d'endocytose médiée par les radeaux lipidiques. Ces résultats suggèrent que le transport de la protéine rapporteur ZEBRA-MD se produit principalement par translocation directe à travers la membrane cellulaire et non par endocytose. La distribution tissulaire d'EGFP ou de la β-galactosidase couplés ou non avec ZEBRA-MD a été étudiée après injection dans la souris. Seules les protéines de fusion avec ZEBRA-MD ont pu être révélées dans les cellules de différents tissus. De plus, la séquence de translocation de ZEBRA-MD a été fusionnée avec la protéine anti-tumorale IL-24/MDA-7. La mort cellulaire induite par l'internalisation de ZEBRA-MD-IL-24/MDA-7 a été mise en évidence par le clivage des caspases de la voie d'apoptose aussi dans des cellules normales que dans des cellules tumorales du sein. En conclusion, le mécanisme d'internalisation de ZEBRA-MD est approprié pour un transport efficace de protéines biologiquement actives

Caractérisation de la propriété de la protéine ZEBRA du virus Epstein-Barr à pénétrer dans les cellules

The basic-leucine zipper (bZIP) transcriptional activator ZEBRA of the Epstein - Barr virus was recently shown to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. During this PhD study, I investigated the potential application of ZEBRA as a transporter protein to facilitate transduction of cargo proteins. The analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization was spanning residues 178-220. The MD efficiently transported reporter proteins, such as EGFP and β-galactosidase, into several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by β-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in presence of heparin. Furthermore, internalization was blocked at 4 °C, whereas no ATP was required as witnessed by an only 25 % decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors had no significant impact on MD-EGFP uptake. Only methyl-β-cyclodextrin (MβCD) inhibited MD-EGFP uptake by 40 % indicating the implication of the lipid raft mediated endocytotic pathway. These data suggest that ZEBRA-MD-reporter protein transduction occurs mostly via direct translocation through the cell membrane and not by endocytosis. Tissue distribution of ZEBRA-MD-EGFP or ZEBRA-MD-β-galactosidase was analyzed after administration into mice. ZEBRA coupled reporter proteins were found in single cells of various tissues contrary to application of EGFP and β-galactosidase alone. In addition, the cell penetrating sequence of ZEBRA (MD) was fused to anti-tumor protein IL-24/MDA-7. Induced cell death after successful internalization of MD-IL-24/MDA-7 was proven by the cleavage of apoptosis specific caspases without showing difference between normal and tumor breast cells. The mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins.Il a été récemment démontré que l'activateur de transcription ZEBRA du virus Epstein-Barr contenant un motif "basic-leucine zipper (bZIP)" traverse la membrane externe des cellules vivantes et s'accumule dans le noyau des lymphocytes. Durant mon travail de thèse, j'ai étudié la possibilité d'utiliser ZEBRA comme protéine de transport afin de faciliter la transduction de protéines cargo. L'analyse de différentes formes tronquées de ZEBRA a permis de mettre en évidence que le domaine minimal (MD) nécessaire à l'internalisation inclut les résidus 178-220. Le MD a permis de transporter de manière efficace des protéines rapporteur comme la EGFP et la -galactosidase dans plusieurs lignées cellulaires normales et tumorales. La fonction des protéines cargo internalisées a été confirmée par l'activité -galactosidase dans les cellules transduites, et aucune toxicité cellulaire associée au MD n'a été détectée. La translocation du MD à travers la membrane cellulaire nécessite la liaison aux héparanes sulfates protéoglycans associés à la surface de la cellule comme cela a été démontré par la forte inhibition du transport de protéines en présence d'héparine. En outre, l'internalisation est également bloquée à basse température (4 °C). De plus, une réduction de seulement 25 % du transport de protéines cargo dans des cellules avec des stocks d'ATP épuisés démontre que l'internalisation est un processus indépendant de l'ATP. Les inhibiteurs classiques d'endocytose n'ont aucun effet significatif sur le transport de MD-EGFP. Seul le methyl--cyclodextrin inhibe de 40 % le transport de MD-EGFP, indiquant l'implication d'une voie d'endocytose médiée par les radeaux lipidiques. Ces résultats suggèrent que le transport de la protéine rapporteur ZEBRA-MD se produit principalement par translocation directe à travers la membrane cellulaire et non par endocytose. La distribution tissulaire d'EGFP ou de la β-galactosidase couplés ou non avec ZEBRA-MD a été étudiée après injection dans la souris. Seules les protéines de fusion avec ZEBRA-MD ont pu être révélées dans les cellules de différents tissus. De plus, la séquence de translocation de ZEBRA-MD a été fusionnée avec la protéine anti-tumorale IL-24/MDA-7. La mort cellulaire induite par l'internalisation de ZEBRA-MD-IL-24/MDA-7 a été mise en évidence par le clivage des caspases de la voie d'apoptose aussi dans des cellules normales que dans des cellules tumorales du sein. En conclusion, le mécanisme d'internalisation de ZEBRA-MD est approprié pour un transport efficace de protéines biologiquement actives

Expression and purification of ZEBRA fusion proteins and applications for the delivery of macromolecules into mammalian cells.

International audienceThe recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution

Expression and Purification of ZEBRA Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells

International audienceThe recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution

Functional characterisation of the WW minimal domain for delivering therapeutic proteins by adenovirus dodecahedron.

International audienceProtein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron) has recently been shown to deliver proteins fused to the tandem WW(2-3-4) structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW(3) and its mutant form WW(3)_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application

Design of WW fusion proteins and their expression.

<p><i>A</i>. GFP fusion constructs were cloned in frame with WW domains from the ubiquitin-ligase Nedd4, including truncated forms containing the shorter linker QGLQNEE (depicted in yellow) and the mutant forms (magnified insert; sequential mutated residues highlighted in red) derived from WW₃ domain, to generate a closely related form to the artificial WW domain CC43 (1). 6-amino acid histidine tag (orange); WW domains (blue barrels); GFP (green barrel). Amino acid length for each domain is indicated. <i>B.</i> WW-GFP fusion proteins were expressed in a cell-free expression system overnight at 20°C and their solubility assessed by Western blot analysis, detected with an anti-histidine HRP antibody. Soluble fraction (red arrows), left; insoluble fraction, right.</p

Structure of Penton-Dodecahedron as protein delivery particle and cellular uptake of WW_2-3-4/Pt-Dd protein complexes in live HeLa cells.

<p><i>A</i>. The Adenovirus type 3 Penton structure is a non covalent complex consisting of the fiber and base protein. The conserved PPxY sequences present in the pentameric base region of Pt-Dd serve as docking regions to bind the structurally conserved WW domains from some ubiquitin ligase proteins. <i>B</i>. Internalisation of Cy3-Pt-Dd (left panel) and Alexa 647- WW_2-3-4 (right panel) in cells measured by FACS analysis. Cells were incubated for 2 h with 1.35 nM (orange histogram), 2.7 nM (green histogram) Cy3-Pt-Dd or 0.1 µM Alexa 647- WW_2-3-4 [internalised by 0.6 nM (cyan histogram) or 1.2 nM (magenta histogram) Pt-Dd]. Non-treated cells, black histogram; cells incubated with Alexa 647- WW_2-3-4 only,grey histogram. <i>C.</i> Internalisation of Cy3-Pt-Dd (signal pseudo-coloured in green for colocalisation purposes) and Alexa 647- WW_2-3-4 (red) in cells measured by fluorescence microscopy. Cells were incubated with 2.7 nM Cy3-Pt-Dd and 0.3µM Alexa 647- WW_2-3-4 for 30 min, washed and further incubated with prewarmed media for 3 h before image acquisition using an Olympus Microscope at a rate of 3 frames per min. Frozen images from the live imaging acquisition (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045416#pone.0045416.s001" target="_blank">Movie S1</a>) showing the internalisation and cellular distribution of Cy3-Pt-Dd (signal pseudo-coloured in green for colocalization purposes), Alexa 647- WW_2-3-4 (red signal) and their merged signals and DIC channel extracted from one picture of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045416#pone.0045416.s001" target="_blank">Movie S1</a>. Nucleus (N) highlighted in white. Vesicle motion during the Olympus filter switch (about 1 s) is highlighted with white arrows in zoomed areas 1 and 2; areas of Dd and WW_2-3-4 colocalization are seen in yellow.</p