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Idar-Oberstein, Germany

Thinon E.,Imperial College London | Thinon E.,Rockefeller University | Serwa R.A.,Imperial College London | Broncel M.,Imperial College London | And 10 more authors.
Nature Communications | Year: 2014

Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. © 2014 Macmillan Publishers Limited. All rights reserved.

Heinz N.,Eufets GmbH | Heinz N.,Hannover Medical School | Hennig K.,Eufets GmbH | Loew R.,Eufets GmbH
BMC Biotechnology | Year: 2013

Background: Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an " All-In-One" vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral " All-In-One" vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.Results: Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose-response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.Conclusion: Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations. © 2013 Niels et al.; licensee BioMed Central Ltd.

Loew R.,Eufets GmbH | Loew R.,University of Heidelberg | Heinz N.,Eufets GmbH | Heinz N.,Hannover Medical School | And 4 more authors.
BMC Biotechnology | Year: 2010

Background: The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state.Results: In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems.Conclusions: Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system. © 2010 Loew et al; licensee BioMed Central Ltd.

Maetzig T.,Hannover Medical School | Galla M.,Hannover Medical School | Brugman M.H.,Hannover Medical School | Loew R.,Eufets GmbH | And 3 more authors.
Gene Therapy | Year: 2010

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O 6-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression. © 2010 Macmillan Publishers Limited All rights reserved.

Boehrer S.,University Paris XIII | Schroeder P.,Eufets GmbH | Mueller T.,Fresenius Biotech GmbH | Atz J.,Fresenius Biotech GmbH | Chow K.U.,Private Practice
Anti-Cancer Drugs | Year: 2011

Monoclonal antibodies such as rituximab and alemtuzumab show considerable therapeutic efficacy in chronic lymphocytic leukaemia (CLL). Aiming to further improve antineoplastic efficacy, the trifunctional bispecific antibody FBTA05 was developed. FBTA05 is thought to function by simultaneously binding B cells and T cells by its variable regions and by recruiting FcγR-positive accessory immune cells by its intact Fc region. As it was previously shown that this antibody shows considerable cytotoxicity towards a spectrum of B-cell lymphoma cell lines, we here tested its potential efficacy ex vivo against malignant B-CLL cells. Therefore, we assessed the capacity of increasing concentrations of FBTA05 to bind to neoplastic cells, to induce cytotoxicity (comparing it with rituximab and alemtuzumab) and cytokine release. We evaluated the results with respect to the extent of CD20 expression, the effector:target cell ratio as well as with the patients' overall effector cell status. Thus, we show that, although FBTA05-elicited cytotoxicity was comparable with that induced by alemtuzumab, it considerably exceeded the antineoplastic effects of rituximab. Noteworthy, FBTA05 shows effective elimination of malignant B cells even if CD20 surface expression is low. Importantly, a high grade of cytotoxicity was associated with the induction of T-cell proliferation and the concomittant release of interferon-γ and interleukin-6, thus overcoming the detrimental effects of an unfavourable effector:target cell ratio. In conclusion, we here present novel evidence for the therapeutic efficacy of the trifunctional, bispecific antibody FBTA05 in CLL and provide evidence for the importance of immune-mediated mechanisms conveying the cytotoxic effects against malignant B lymphocytes. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

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