Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers.

OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium's natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located in the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.

Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems.

The mitochondrial voltage-dependent anion channel (VDAC) is a pivotal protein since it provides the major transport pathway between the cytosol and the mitochondrial intermembrane space and it is implicated in cell apoptosis by functioning as a gatekeeper for the trafficking of mitochondrial death molecules. VDAC is a beta-barrel channel with a large conductance, and we use it as a model transport protein for the design of biomimetic systems. To overcome the limitations of classical overexpression methods for producing and purifying membrane proteins (MPs) we describe here the use of an optimized cell-free system. In a one-step reaction VDAC is obtained directly integrated into liposomes and purified by ultracentrifugation. We then combine proteoliposomes with different bilayers models in order to validate VDAC insertion and functionality. This VDAC biomimetic model is the first example validating the use of a cell-free expression system for production of MPs into liposomes and tethered bilayers as a toolbox to build a wide range of biomimetic devices.

Anti-Tumor Effects of Bak-Proteoliposomes against Glioblastoma.

Despite palliative treatments, glioblastoma (GBM) remains a devastating malignancy with a mean survival of about 15 months after diagnosis. Programmed cell-death is de-regulated in almost all GBM and the re-activation of the mitochondrial apoptotic pathway through exogenous bioactive proteins may represent a powerful therapeutic tool to treat multidrug resistant GBM. We have reported that human Bak protein integrated in Liposomes (LB) was able, in vitro, to activate the mitochondrial apoptotic pathway in colon cancer cells. To evaluate the anti-tumor effects of LB on GBM, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and Western blot analysis were performed on GL26 murine cell line. LB treatment shows a dose-dependent inhibition of cell viability, followed by an up-regulation of Bax and a down-modulation of JNK1 proteins. In GL26-bearing mice, two different routes of administration were tested: intra-tumor and intravenous. Biodistribution, tumor growth and animal survival rates were followed. LB show long-lasting tumor accumulation. Moreover, the intra-tumor administration of LB induces tumor growth delay and total tumor regression in about 40% of treated mice, while the intravenous injection leads to a significant increased life span of mice paralleled by an increased tumor cells apoptosis. Our findings are functional to the design of LB with potentiated therapeutic efficacy for GBM.

Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator.

The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the Epstein-Barr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170-220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and beta-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by beta-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 the presence of heparin. We found that internalization was blocked at 4 degrees C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-beta-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.

Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes.

Pseudomonas aeruginosa is the second-leading cause of nosocomial infections and pneumonia in hospitals. Because of its extraordinary capacity for developing resistance to antibiotics, treating infections by Pseudomonas is becoming a challenge, lengthening hospital stays, and increasing medical costs and mortality. The outer membrane protein OprF is a well-conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. Here, we used a bacterial cell-free expression system to reconstitute OprF under its native forms in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa Remarkably, we showed that our system promotes the folding of OprF into its active open oligomerized state as well as the formation of mega-pores. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.

Single-step production of functional OEP24 proteoliposomes.

The pea chloroplastic outer envelope protein OEP24 is a voltage-dependent channel that can function as a general solute channel in plants. OEP24 is a close functional homologue of VDAC which, in mammalian cells, modulates the permeability of the outer mitochondrial membrane. Here, we describe the production in a one-step reaction of active OEP24 in proteoliposomes or in soluble form using a cell-free expression system. We combine evidence from electrophysiological experiments, biophysical characterization, and biochemical analysis demonstrating that OEP24 is present as a functional channel in liposomes. Thus, production of OEP-containing proteoliposomes may provide a helpful tool for deciphering the role of the OEP family members.

Production of membrane proteins using cell-free expression systems.

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane ß-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway.

A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models.

We discuss the current challenges and future prospects of flow-based organoid models and 3D self-assembling scaffolds. The existing paradigm of 3D culture suffers from a lack of control over organoid size and shape; can be an obstacle for cell harvesting and extended cellular and molecular analysis; and does not provide access to the function of exocrine glands. Moreover, existing organ-on-chip models are mostly composed of 2D extracellular matrix (ECM)-coated elastomeric membranes that do not mimic real organ architectures. A new comprehensive 3D toolbox for cell biology has emerged to address some of these issues. Advances in microfabrication and cell-culturing approaches enable the engineering of sophisticated models that mimic organ 3D architectures and physiological conditions, while supporting flow-based drug screening and secretomics-based diagnosis.

Inhibition of cell growth and induction of apoptosis in human prostate cancer cell lines by 6-aminoquinolone WM13.

Fluoroquinolones affect the proliferation and apoptotic cell death of several human malignancies. Therefore, we investigated whether new 6-aminoquinolone derivatives, initially synthesized as anti-HIV agents, could affect the proliferation and apoptotic cell death of human prostate cancer cell lines. PC3 and LNCaP cell lines were used as models of androgen-resistant and androgen-responsive prostate cancer, and proliferation of PC3 and LNCaP cells was strongly inhibited by 6-aminoquinolone WM13. Cytotoxicity, which was more pronounced in LNCaP, was accompanied by morphological changes, DNA damage, arrest at the S/G(2)/M phase of the cell cycle, and an increase of the sub-G(1) population. Molecular mechanism underlying WM13-induced cell death involved caspase-8 and -3 and modulation of the expression of apoptotic genes, as well as cleavage of poly-ADP ribose polymerase. Cell death following the treatment of human prostate cancer cell lines with WM13 can be attributed to apoptosis which, depending on the cell line, proceeds through different pathways.

MD11-mediated delivery of recombinant eIF3f induces melanoma and colorectal carcinoma cell death.

The f subunit of the eukaryotic initiation factor 3 (eIF3f) is downregulated in several cancers and in particular in melanoma and pancreatic cancer cells. Its enforced expression by transient gene transfection negatively regulates cancer cell growth by activating apoptosis. With the aim to increase the intracellular level of eIF3f proteins and activate apoptosis in cancer cell lines, we developed a protein transfer system composed of a cell-penetrating peptide sequence fused to eIF3f protein sequence (MD11-eIF3f). To determine whether exogenously administered eIF3f proteins were able to compensate the loss of endogenous eIF3f and induce cancer cell death, we analyzed the therapeutic action of MD11-eIF3f in several tumor cells. We identified four cell lines respondent to eIF3f-treatment and we evaluated the antitumor properties of the recombinant proteins using dose- and time-dependent studies. Our results demonstrate that this protein delivery approach represents an innovative and powerful strategy for cancer treatment.

Electron microscopic cytochemistry of adenylyl cyclase activity in mouse spermatozoa.

We investigated adenylyl cyclase activity of mouse spermatozoa by electron microscopic cytochemistry. Subcellular localization of enzyme activity was determined in the presence and absence of bicarbonate ions. Results confirm the existence in sperm of a bicarbonate-regulated adenylyl cyclase, which suggests microdomain signaling.

Involvement of A1 adenosine receptors in the acquisition of fertilizing capacity.

Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two-step process: capacitation followed by acrosome reaction. The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A(1) receptor (A(1)R). Mice with a targeted disruption of the Adora 1 gene (A(1)R-/- mice) provide a useful model for better understanding the role of the A(1)R in fertility. Murine spermatozoa express A(1)R in the head, neck, midpiece region, and tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A(1)R-/- compared with A(1)R+/+ and A(1)R+/- spermatozoa. The difference between A(1) R+/+ and A(1)R-/- mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A(1)R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A(1)R-/- sperm required 240 minutes. Caffeine, a known antagonist of A(1) and A(2A) adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose-dependent manner, mimicking the effects of the lack of A(1) receptors. Although number, motility, and viability of A(1)R-/- murine sperm was not significantly different from A(1)R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A(1)R-/- male mice suggests that A(1) receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.

Raman micro-spectroscopy: a powerful tool for the monitoring of dynamic supramolecular changes in living cells.

Cellular imaging techniques have become powerful tools in cell biology. With respect to others, the techniques based on vibrational spectroscopy present a clear advantage: the molecular composition and the modification of subcellular compartments can be obtained in label-free conditions. In fact, from the evolution of positions, intensities and line widths of Raman and infrared bands in the cell spectra, characteristic information on cellular activities can be achieved, and particularly, cellular death can be investigated. In this work we present the time evolution of the Raman spectra of single live Jurkat cells (T-lymphocyte) by looking at the high frequency part of their Raman spectra, that is the CH stretching region, around 3000cm(-1). In particular, investigation into the composition or rearrangement of CH bounds, markers of cellular membrane fatty acids, can represent an important method to study and to recognize cell death. The experimental procedure we used, together with the analysis of these high frequency vibrational bands, may represent a new, improved and advantageous approach to this kind of study.

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

Protein 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.

Production of recombinant proteoliposomes for therapeutic uses.

One of the major challenges in human therapy is to develop delivery systems that are convenient and effective for tackling problems in disease treatments. In the past 20 years, liposomes have represented promising pharmaceutical carriers for drug delivery. Due to their biophysical properties, liposomes can deliver and specifically target a large set of bioactive molecules, they can protect molecules from degradation, and their composition is easily modifiable. The use of recombinant proteoliposomes containing therapeutic membrane proteins is a recently developed technology that allows biologically active proteins to penetrate across the plasma membrane of eukaryotic cells. One of the bottlenecks in this powerful delivery system lies in the production of functional therapeutic membrane proteins mainly due to their biophysical characteristics. Membrane proteins represent about 30% of the total proteins from an organism, and play a central role in drug discovery as potential pharmaceutical targets. This chapter describes the methodology for the production of bioactive proteoliposomes containing therapeutic, proapoptotic membrane proteins synthesized with an optimized cell-free expression system. We will examine (1) the design of the expression vectors and the liposome compositions compatible with the cell-free expression system; (2) the production of membrane proteins using a cell-free expression system in combination with liposomes, to obtain in a one-step reaction functional therapeutic proteoliposomes; (3) proteoliposome purification for further use in the treatment of cancer cells; and (4) the methodology for detecting apoptosis in cells after treatment. Furthermore, this system can be easily adapted for producing "difficult to express proteins" compared with the classical overexpression (bacterial or eukaryotic) systems.

A bacterial cell-free expression system to produce membrane proteins and proteoliposomes: from cDNA to functional assay.

Limitations in the production of folded membrane proteins represent the major bottleneck for functional and structural studies of this huge category of macromolecules. Cell-free expression systems provide an attractive alternative to the classical overexpression systems for producing membrane proteins. However, optimization of these systems remains a challenging task, considering the hydrophobic properties of these molecules. This unit describes the production of eukaryotic membrane proteins either in soluble form or integrated into liposomes using a bacterial cell-free expression system. Liposomes in the reaction mixture induce the direct insertion of freshly produced membrane proteins into the bilayer and allow the formation of functional proteoliposomes in which the membrane proteins are correctly folded.

Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins.

The delivery of functional therapeutic proteins by lipid vesicles into targeted living cells is one of the most promising strategies for treatment of different diseases and cancer. The use of this system in the delivery of membrane proteins directly into cells remains to be tested because the methods for producing membrane proteins are difficult to perform. Here we describe the effect of proteoliposomes containing the voltage-dependent anion channel (VDAC) and pro-apoptotic Bak, both produced with an optimized cell-free expression system. For the first time, recombinant VDAC and Bak proteins are synthesized and directly integrated into the lipidic bilayer of natural liposomes in a one-step reaction. VDAC has been shown to play an essential role in apoptosis in mammalian cells by regulating cytochrome c release from mitochondria and Bak modulates mitochondrial membrane permeability upon activation. Internalization of recombinant proteoliposomes into mammalian cells induces apoptosis by release of cytochrome c and caspases activation. These results highlight that membrane proteins integrated in natural liposomes can represent an excellent candidate for cancer protein therapy.

Liposome-mediated cellular delivery of active gp91(phox).

BACKGROUND: 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.