Mostafa Bentahir

Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.

An Influenza A Vaccine Based on Tetrameric Ectodomain of Matrix Protein 2

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzyme-linked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.

Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species.

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated from Antarctica has been purified and characterized. The gene encoding PAP has been cloned and sequenced and the derived amino acid sequence shows 66% identity with the mesophilic alkaline metalloprotease from Pseudomonas aeruginosa IFO 3455 (AP). Compared to the purified AP, PAP is three times more active at 20 degrees C, is very sensitive to chelating agents and is rapidly inactivated at 45 degrees C. The lower thermostability of PAP can tentatively be explained by a loss of a stabilizing Ca(2+), a decrease in the content of hydrophobic residues and a smaller aliphatic index.

Cold-Adapted Enzymes

In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

Hierarchical and redundant roles of activating FcγRs in protection against influenza disease by M2e-specific IgG1 and IgG2a antibodies

ABSTRACT The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g−/−, Fcgr3 −/−, and Fcgr1 −/− Fcgr3 −/− BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies. IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Rho GTPase Cdc42 is essential for human T-cell development.

Background Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored. Design and Methods We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration. Results We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34+ fraction, towards stromal cell-derived factor-1α. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1α and T-cell development to different degrees. Conclusions This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.

Crystallization and preliminary X-ray analysis of a bacterial psychrophilic enzyme, phosphoglycerate kinase

The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.

New host factors and pathways involved in CD4 downregulation in HIV-1 infected cells

Background: Downregulation of the CD4 receptor is one of the hallmarks of HIV infection. The virus has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation, mainly mediated by three viral proteins: Vpu, Env and Nef. We were interested in the discovery of pathways and human proteins involved in the process, which eventually could represent new drug targets. Materials and methods: A genome-wide short-hairpin RNA (shRNA) screening using a SBI shRNA lentiviral interference delivery system library compatible with the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix) was performed in HeLa CD4+ cells expressing the Nef protein introduced by retroviral transduction. CD4 surface levels were measured by flow cytometry. The read-out in the screen showed the rescue of the CD4-high phenotype despite Nef expression. shRNA sequences enriched in the CD4-high cells compared to the CD4-low cells were identified and filtered via pathway analysis. For the confirmation and further selection of the hits two cell lines in different conditions were used: 1. HeLa CD4+ cells expressing Nef after retroviral transduction (similar to previous screening effort). 2. SupT1 lymphocytic cells infected with replication competent HIV-1 encoding a GFP reporter. 3. SupT1 cells expressing Nef or Vpu after retroviral transduction. In all three experimental set-ups, the cells were selectively knocked-down for each of the hits individually after transduction with a different set of shRNA encoding lentiviral vectors (Mission Consortium, Sigma Aldrich) prior to HIV-1 infection or retroviral transduction. Results: The genome-wide screen with the SBI library was repeated 4 times to obtain a final list of 75 genes as a first selection of possible new host co-factors in CD4 downregulation by Nef. Of these, 22 proteins were confirmed independently with individual Mission consortium vectors in the same cell line. Eight proteins contributed to CD4 downregulation in HIV-1 infected SupT1 cells. The host factors identified show differential effect on CD4 surface levels in SupT1 cells expressing either HIV-1 Vpu or Nef proteins individually, that together determine CD4 levels on infected cells. These proteins are mainly involved in endosomal and trans Golgi network (TGN) trafficking. Conclusion: Several host proteins involved in endosomal and TGN trafficking differentially affect Nef or Vpu mediated CD4 down-regulation in HIV1-1 infected cells.

Use of RNA interference to discover pathways involved in HIV infection and replication: cell lines tell many stories, primary cells might tell the truth

Infection by Human Immunodeficiency Virus is difficult to treat thanks to its persistent viral reservoir and to its high rate of mutation that allows appearance of resistance to the available treatment. An approach to discover drugable targets is the identification of cellular partner proteins interacting with the virus during its life cycle. We used RNAi technology to identify new HIV partners in the host cytoskeleton since it has been shown that cytoskeleton components and the irregulators have a role during several steps of HIV life cycle. By transducingseveral Tcell lines such as Jurkat CD4 CCR5, Jurkat E6-1 and SupT1 with lentiviral vectors expressings hRNA sequences, we silenced different target genes, members of pathways involved inactinrearrangement. By infection with HIV-NL4.3-eGFP reporter virus we evaluated HIV replication ratesin transducedcells. Surprisingly, the infection rate affected by the specific knock-down was dependent on the cell line used. lndeed, ashRNAtransduced in one cell line affected infection differently to what it did in another. Moreover, we observed that transduction on itself with a control vector expressinga scrambled shRNA sequence affected HIV infection rate in some but not all cell lines. Therefore, to obtain relevant results in screening co-factors for HIV infection, we turned toprimary cells, the naturaltargets of the virus in vivo. We optimized combinedlentiviral transduction and HIV infection in cultured peripheral blood CD4+ lymphocytes. In this setting, transduction with scrambled shRNA expressing lentivirus did not affect HIV replication, providing us a platform to assay gene-knockdown likely to generate the most relevant information for natural HIV infection invivo.

Evaluation of pathways and new host proteins involved in CD4 down-modulation during HIV-1 infection

Down-modulation of the CD4 receptor expressed on T cells is one of the hallmarks of Human Immunodeficiency Virus (HIV) infection and it is believed to confer to the virus a selective replicative advantage in vivo. HIV has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation. This process is mainly mediated by three viral proteins: Vpu, Env and Nef. Up to date, the mechanisms that lead to CD4 depletion from the surface of CD4+ T lymphocytes, the natural targets of HIV, are still poorly understood and only partially characterized. We are interested in the discovery of pathways and human proteins involved in the process, in order to eventually find potential new drug targets. To pursue our aim, we first performed a functional screening on HeLa CD4+ cells expressing Nef, using a shRNA lentiviral interference delivery system targeting the whole human genome compatible with an Affymetrix GeneChip Microarray. The read out was the rescue of the CD4 high phenotype, despite Nef expression. The results were analyzed with Affymetrix Expression Console 1.1 software and the web-based gene ontology resource DAVID. After four different screens we obtained a final list of 75 genes enriched in the cells sorted for high surface CD4. These genes appear to code for proteins involved in endocytic trafficking, trans-Golgi trafficking and lysosomal degradation pathways. We decided to validate our hits in primary blood CD4+ T cells, the most relevant for this kind of studies, and we set up a protocol for the transduction of these cells with lentiviral and retroviral vectors, necessary respectively for the expression of the shRN As and the viral proteins Vpu, Nef and Env. The actual down-regulation of the gene expression will be validated via quantitative Real Time PCR and Western Blot and the experiments will be eventually repeated in the context of HIV-1 infection.