RuX: A Novel, Flexible, and Sensitive Mifepristone-Induced Transcriptional Regulation System.

Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system, was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures.

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts.

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

Placental Galectins Are Key Players in Regulating the Maternal Adaptive Immune Response.

Galectins are potent immunomodulators that regulate maternal immune responses in pregnancy and prevent the rejection of the semi-allogeneic fetus that also occurs in miscarriages. We previously identified a gene cluster on Chromosome 19 that expresses a subfamily of galectins, including galectin-13 (Gal-13) and galectin-14 (Gal-14), which emerged in anthropoid primates. These galectins are expressed only by the placenta and induce the apoptosis of activated T lymphocytes, possibly contributing to a shifted maternal immune balance in pregnancy. The placental expression of Gal-13 and Gal-14 is decreased in preeclampsia, a life-threatening obstetrical syndrome partly attributed to maternal anti-fetal rejection. This study is aimed at revealing the effects of Gal-13 and Gal-14 on T cell functions and comparing the expression of these galectins in placentas from healthy pregnancies and miscarriages. First-trimester placentas were collected from miscarriages and elective termination of pregnancies, tissue microarrays were constructed, and then the expression of Gal-13 and Gal-14 was analyzed by immunohistochemistry and immunoscoring. Recombinant Gal-13 and Gal-14 were expressed and purified, and their effects were investigated on primary peripheral blood T cells. The binding of Gal-13 and Gal-14 to T cells and the effects of these galectins on apoptosis, activation marker (CD25, CD71, CD95, HLA-DR) expression and cytokine (IL-1ß, IL-6, IL-8, IL-10, IFNγ) production of T cells were examined by flow cytometry. Gal-13 and Gal-14 are primarily expressed by the syncytiotrophoblast at the maternal-fetal interface in the first trimester, and their placental expression is decreased in miscarriages compared to first-trimester controls. Recombinant Gal-13 and Gal-14 bind to T cells in a population- and activation-dependent manner. Gal-13 and Gal-14 induce apoptosis of Th and Tc cell populations, regardless of their activation status. Out of the investigated activation markers, Gal-14 decreases the cell surface expression of CD71, Gal-13 increases the expression of CD25, and both galectins increase the expression of CD95 on T cells. Non-activated T cells produce larger amounts of IL-8 in the presence of Gal-13 or Gal-14. In conclusion, these results show that Gal-13 and Gal-14 already provide an immunoprivileged environment at the maternal-fetal interface during early pregnancy, and their reduced expression is related to miscarriages.

Cutting Edge: A New Player in the Alternative Complement Pathway, MASP-1 Is Essential for LPS-Induced, but Not for Zymosan-Induced, Alternative Pathway Activation.

The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding-associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.

Dissociation and re-association studies on the interaction domains of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, provide evidence for heterodimer formation.

Activation of the lectin pathway of complement begins with the activation of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, which are bound to the recognition molecules, MBL and ficolins. MASPs are Ca(2+)-dependent dimers. Dimerization and Ca(2+)-dependent association with the recognition molecules occurs via the first 3 domains, the CUB1-EGF-CUB2 region. The CUB1-EGF-CUB2 (D1-3) regions of MASP-1 and MASP-2, and also their tagged versions, were expressed in E. coli, refolded and purified. The first three domains of MASP-1 are identical with the respective regions of MASP-3 and MAp44, which are also associated with MBL and ficolins. The functionality of the fragments was checked by inhibition of C3 deposition from human serum. Time-course of the dissociation and re-association was examined by size exclusion chromatography. Both refolded proteins are tight Ca(2+)-dependent dimers, as expected. In buffer containing EDTA MASP-1_D1-3 dissociated to monomers, however it took about 1h to reach an equilibrium. Upon re-calcification dimers were re-formed, but this process was even slower; only after overnight incubation was the dimerization completed. MASP-2_D1-3 showed a somewhat different behavior: dissociation by EDTA was even slower, less complete, and higher MW aggregates also appeared. Heterodimer formation was detected by native PAGE. As modeled by the D1-3 fragments, MASP-1 and MASP-2 can readily form heterodimers after dissociation and re-association, however, in the presence of Ca(2+) exchange of subunits is slow between the homodimers. MASP-1:MASP-3 heterodimer formation was modeled by the tagged and untagged D1-3 fragments, and data indicate that subunits of these proteins are readily exchanged even in the presence of Ca(2+). The existence of heterodimers influences the current view on the composition of lectin pathway complexes and their activation.

The control of the complement lectin pathway activation revisited: both C1-inhibitor and antithrombin are likely physiological inhibitors, while α2-macroglobulin is not.

The lectin pathway of complement is an important effector arm of innate immunity. It forms a first line of defense against invading pathogens and dangerously altered self structures. Pattern recognition molecules (mannose-binding lectin (MBL), ficolins) bind to the dangerous particles, which is followed by activation of MBL-associated serine proteases, MASP-1 and MASP-2, resulting in the initiation of the complement cascade. The activation of the lectin pathway is strictly controlled by natural inhibitors, since uncontrolled activation can lead to serious self-tissue damage. Recently we have shown that inhibition of either MASP-1 or MASP-2 by in vitro evolved specific inhibitors completely blocks the lectin pathway in human serum. In this study, we examined the inhibitory action of C1-inhibitor (C1-inh), antithrombin (AT) and α(2)-macroglobulin (α(2)M) on MASP-1 and MASP-2, and studied the inhibition of the lectin pathway in normal human serum in the presence and absence of heparin using C3 and C4 deposition assays. We measured the association rate constants for the serpin/protease reactions. We found that in the presence of heparin both C1-inh and AT are equally efficient inhibitors of the lectin pathway. Although α(2)M formed complex with MASP-1 in fluid phase, it could not abolish lectin pathway activation on activator surfaces.

CR3 is the dominant phagocytotic complement receptor on human dendritic cells.

Dendritic cells (DCs) play a decisive role in immunity; they interact with various pathogens via several pattern recognition and different opsonophagocytotic receptors, including Fc- and complement-receptors. ß2-integrins, including complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) participate in many immunological processes, especially those involving cell migration, adherence, and phagocytosis. Human monocyte derived dendritic cells (MDCs) are known to express CR3 as well as CR4, however possible differences regarding the role of these receptors has not been addressed so far. Our aim was to explore whether there is a difference between the binding and uptake of various complement-opsonized microorganisms, mediated by CR3 and CR4. Studying the expression of receptors during differentiation of MDCs we found that the appearance of CD11b decreased, whereas that of CD11c increased. Interestingly, both receptors were present in the cell membrane in an active conformation. Here we demonstrate that ligation of CD11b directs MDCs to enhanced phagocytosis, while the maturation of the cells and their inflammatory cytokine production are not affected. Blocking CD11c alone did not change the uptake of opsonized yeast or bacteria by MDCs. We confirmed these results using siRNA; namely downregulation of CD11b blocked the phagocytosis of microbes while silencing CD11c had no effect on their uptake. Our data clearly demonstrate that complement C3-dependent phagocytosis of MDCs is mediated mainly by CR3.