Publications by authors named "Robbert H Cool"

40 Publications

D-dopachrome tautomerase contributes to lung epithelial repair via atypical chemokine receptor 3-dependent Akt signaling.

EBioMedicine 2021 Jun 4;68:103412. Epub 2021 Jun 4.

Groningen Research Institute of Pharmacy, Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands; University Medical Center Groningen, Groningen Research Institute of Asthma and COPD, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. Electronic address:

Background: Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD.

Methods: We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation.

Findings: We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells.

Interpretation: In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD.

Funding: As stated in the Acknowledgments.
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http://dx.doi.org/10.1016/j.ebiom.2021.103412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185224PMC
June 2021

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Angew Chem Int Ed Engl 2021 08 26;60(32):17514-17521. Epub 2021 Jun 26.

Department Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.
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http://dx.doi.org/10.1002/anie.202101864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362126PMC
August 2021

7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

J Med Chem 2020 10 13;63(20):11920-11933. Epub 2020 Oct 13.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site ( = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586407PMC
October 2020

Heightened JNK Activation and Reduced XIAP Levels Promote TRAIL and Sunitinib-Mediated Apoptosis in Colon Cancer Models.

Cancers (Basel) 2019 Jun 26;11(7). Epub 2019 Jun 26.

Department of Medicine, University of Arizona Cancer Center, Tucson, AZ 85724, USA.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib and would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.
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http://dx.doi.org/10.3390/cancers11070895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678293PMC
June 2019

Creation of RANKL mutants with low affinity for decoy receptor OPG and their potential anti-fibrosis activity.

FEBS J 2019 09 25;286(18):3582-3593. Epub 2019 May 25.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.
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http://dx.doi.org/10.1111/febs.14925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852375PMC
September 2019

Small-molecule inhibitors of macrophage migration inhibitory factor (MIF) as an emerging class of therapeutics for immune disorders.

Drug Discov Today 2018 11 21;23(11):1910-1918. Epub 2018 Jun 21.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is an important cytokine for which an increasing number of functions is being described in the pathogenesis of inflammation and cancer. Nevertheless, the availability of potent and druglike MIF inhibitors that are well-characterized in relevant disease models remains limited. Development of highly potent and selective small-molecule MIF inhibitors and validation of their use in relevant disease models will advance drug discovery. In this review, we provide an overview of recent advances in the identification of MIF as a pharmacological target in the pathogenesis of inflammatory diseases and cancer. We also give an overview of the current developments in the discovery and design of small-molecule MIF inhibitors and define future aims in this field.
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http://dx.doi.org/10.1016/j.drudis.2018.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453109PMC
November 2018

High yield production of human invariant chain CD74 constructs fused to solubility-enhancing peptides and characterization of their MIF-binding capacities.

Protein Expr Purif 2018 08 27;148:46-53. Epub 2018 Mar 27.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands. Electronic address:

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.
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http://dx.doi.org/10.1016/j.pep.2018.03.008DOI Listing
August 2018

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor.

Bioorg Med Chem 2018 03 24;26(5):999-1005. Epub 2017 Dec 24.

Department of Chemical and Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC's in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.
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http://dx.doi.org/10.1016/j.bmc.2017.12.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323139PMC
March 2018

Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis.

FEBS J 2017 08 7;284(15):2501-2512. Epub 2017 Jul 7.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

Bone is a dynamic tissue that is maintained by continuous renewal. An imbalance in bone resorption and bone formation can lead to a range of disorders, such as osteoporosis. The receptor activator of NF-κB (RANK)-RANK-ligand (RANKL) pathway plays a major role in bone remodeling. Here, we investigated the effect of mutations at position I248 in the DE-loop of murine RANKL on the interaction of RANKL with RANK, and subsequent activation of osteoclastogenesis. Two single mutants, RANKL I248Y and I248K, were found to maintain binding and have the ability to reduce wild-type RANKL-induced osteoclastogenesis. The generation of RANK-antagonists is a promising strategy for the exploration of new therapeutics against osteoporosis.
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http://dx.doi.org/10.1111/febs.14142DOI Listing
August 2017

Receptor-specific TRAIL as a means to achieve targeted elimination of activated hepatic stellate cells.

J Drug Target 2017 04 28;25(4):360-369. Epub 2016 Dec 28.

b Department of Chemical and Pharmaceutical Biology , University of Groningen , Groningen , the Netherlands.

Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.
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http://dx.doi.org/10.1080/1061186X.2016.1262867DOI Listing
April 2017

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

Cancer Biol Ther 2014 ;15(12):1658-66

a National University of Ireland; Galway; National Centre for Biomedical Engineering Science and Apoptosis Research Centre; Molecular Therapeutics Group ; Galway , Ireland.

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.
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http://dx.doi.org/10.4161/15384047.2014.972183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623120PMC
September 2015

Deinococcus radiodurans can interfere with quorum sensing by producing an AHL-acylase and an AHL-lactonase.

FEMS Microbiol Lett 2014 Jul 26;356(1):62-70. Epub 2014 Jun 26.

Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands.

Bacterial communication via the secretion of small diffusible compounds allows microorganisms to regulate gene expression in a coordinated manner. As many virulence traits are regulated in this fashion, disruption of chemical communication has been proposed as novel antimicrobial therapy. Quorum-quenching enzymes have been a promising discovery in this field as they interfere with the communication of Gram-negative bacteria. AHL-lactonases and AHL-acylases have been described in a variety of bacterial strains; however, usually only one of these two groups of enzymes has been described in a single species. We report here the presence of a member of each group of enzymes in the extremophile bacterium Deinococcus radiodurans. Co-occurrence of both enzymes in a single species increases the chance of inactivating foreign AHL signals under different conditions. We demonstrate that both enzymes are able to degrade the quorum-sensing molecules of various pathogens subsequently affecting virulence gene expression. These studies add the quorum-quenching enzymes of D. radiodurans to the list of potent quorum-quenchers and highlight the idea that quorum quenching could have evolved in some bacteria as a strategy to gain a competitive advantage by altering gene expression in other species.
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http://dx.doi.org/10.1111/1574-6968.12479DOI Listing
July 2014

Assessing Pseudomonas virulence with nonmammalian host: Galleria mellonella.

Methods Mol Biol 2014 ;1149:681-8

Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands.

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for severe to deadly infections in patients suffering from cystic fibrosis, AIDS, undergoing immune suppressing therapies or suffering from severe burns. In the recent years there has been an increasing interest in exploring animal infection models that, to a certain extent, could mimic human infections. Here we describe the use of the larvae of the greater wax moth Galleria mellonella as a non-expensive, easy-to-use, and easy-to-obtain animal model to study P. aeruginosa infections.
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http://dx.doi.org/10.1007/978-1-4939-0473-0_52DOI Listing
March 2015

PvdP is a tyrosinase that drives maturation of the pyoverdine chromophore in Pseudomonas aeruginosa.

J Bacteriol 2014 Jul 9;196(14):2681-90. Epub 2014 May 9.

Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan, Groningen, The Netherlands

The iron binding siderophore pyoverdine constitutes a major adaptive factor contributing to both virulence and survival in fluorescent pseudomonads. For decades, pyoverdine production has allowed the identification and classification of fluorescent and nonfluorescent pseudomonads. Here, we demonstrate that PvdP, a periplasmic enzyme of previously unknown function, is a tyrosinase required for the maturation of the pyoverdine chromophore in Pseudomonas aeruginosa. PvdP converts the nonfluorescent ferribactin, containing two iron binding groups, into a fluorescent pyoverdine, forming a strong hexadentate complex with ferrous iron, by three consecutive oxidation steps. PvdP represents the first characterized member of a small family of tyrosinases present in fluorescent pseudomonads that are required for siderophore maturation and are capable of acting on large peptidic substrates.
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http://dx.doi.org/10.1128/JB.01376-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097581PMC
July 2014

Reducing virulence of the human pathogen Burkholderia by altering the substrate specificity of the quorum-quenching acylase PvdQ.

Proc Natl Acad Sci U S A 2014 Jan 13;111(4):1568-73. Epub 2014 Jan 13.

Department of Pharmaceutical Biology, University of Groningen, 9713 AV, Groningen, The Netherlands.

The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQ(Lα146W,Fβ24Y) conferred high activity toward C8-HSL. Exogenous addition of PvdQ(Lα146W,Fβ24Y) dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition, we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific bacterial species inside a complex microbial community.
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http://dx.doi.org/10.1073/pnas.1311263111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910591PMC
January 2014

The multiple signaling systems regulating virulence in Pseudomonas aeruginosa.

Microbiol Mol Biol Rev 2012 Mar;76(1):46-65

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.
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http://dx.doi.org/10.1128/MMBR.05007-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424PMC
March 2012

Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

FEMS Microbiol Lett 2012 Apr 24;329(2):146-53. Epub 2012 Feb 24.

Department of Pharmaceutical Biology, University Center of Pharmacy, Groningen University Institute for Drug Exploration, Groningen, The Netherlands.

Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.
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http://dx.doi.org/10.1111/j.1574-6968.2012.02516.xDOI Listing
April 2012

Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by receptor specificity: apoptosis induction by TRAIL.

Mol Cell Proteomics 2012 Mar 2;11(3):M111.013730. Epub 2012 Jan 2.

School of Natural Sciences, National University of Ireland, Galway, Ireland.

Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.
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http://dx.doi.org/10.1074/mcp.M111.013730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3316727PMC
March 2012

PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily.

Microbiology (Reading) 2011 Jul 3;157(Pt 7):2042-2055. Epub 2011 Mar 3.

Department of Pharmaceutical Biology, University of Groningen, 9713AV Groningen, The Netherlands.

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like β-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and β-subunits, with the catalytic serine as the first amino acid of the β-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.
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http://dx.doi.org/10.1099/mic.0.043935-0DOI Listing
July 2011

Targeting AML through DR4 with a novel variant of rhTRAIL.

J Cell Mol Med 2011 Oct;15(10):2216-31

Department of Biochemistry and the National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.
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http://dx.doi.org/10.1111/j.1582-4934.2010.01211.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394230PMC
October 2011

Unraveling the binding mechanism of trivalent tumor necrosis factor ligands and their receptors.

Mol Cell Proteomics 2011 Jan 17;10(1):M110.002808. Epub 2010 Sep 17.

Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Characterization of the binding of a tumor necrosis factor (TNF) ligand to its receptor(s) is pivotal to understand how these proteins initiate signal transduction pathways. Unfortunately, kinetic elucidation of these interactions is strongly hampered by the multivalent nature of the binding partners. The interaction between TNF-related apoptosis-inducing ligand and its death receptors was analyzed using in-depth applications of surface plasmon resonance technology. Variations in receptor density and sensor chip type allowed us to manipulate the stoichiometry of the formed complex, and the rate constants describing the binding of trimeric TNF-related apoptosis-inducing ligand to only one receptor molecule were determined. Remarkably, the affinity of this trimer-monomer complex is in the picomolar range, and its dissociation very slow. Further analysis showed that the second and third receptor molecules bind with lower affinity to the preformed trimer-monomer complex. This together with results obtained with receptor activator of NF-κB ligand and B cell-activating factor strongly suggests that the binding of TNF family ligands to their receptors is initiated via the formation of a trimer-monomer complex that is sufficiently stable to allow binding of two additional receptor molecules. These results suggest that avidity does not play a significant role and thus provide new insight in how TNF ligands form the biologically important complexes with their receptors.
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http://dx.doi.org/10.1074/mcp.M110.002808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013454PMC
January 2011

The acylase PvdQ has a conserved function among fluorescent Pseudomonas spp.

Environ Microbiol Rep 2010 Jun 30;2(3):433-9. Epub 2010 Mar 30.

Department of Pharmaceutical Biology, 9713AV Groningen, the Netherlands. School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK.

Pyoverdine biosynthesis in fluorescent Pseudomonas spp. and especially in the opportunistic human pathogen Pseudomonas aeruginosa has been extensively studied. The acylase PvdQ is required for a maturation step in pyoverdine biosynthesis but also has been proven to be effective in degrading long-chain N-acyl homoserine lactones (AHLs). These molecules are used as quorum-sensing molecules by Gram-negative bacteria such as Pseudomonads themselves. Interestingly, the pvdQ gene is part of a pyoverdine cluster in P. aeruginosa and P. syringae but not in other fluorescent Pseudomonas spp. In this study we have compared the activities of PvdQ orthologues from various species and provide evidence for conserved functions in Pseudomonas fluorescens PfO-1, P. putida KT2440 and P. aeruginosa PA14. Despite large differences in genomic organization, expression of each of these pvdQ orthologues is regulated by iron availability. Moreover, PvdQ and its orthologues have conserved substrate specificity for AHLs and play a role in pyoverdine production in all tested Pseudomonas species. These data strongly suggest that the role of PvdQ in pyoverdine biosynthesis is conserved among Pseudomonas spp., while the control that PvdQ exerts in P. aeruginosa over its own quorum-sensing signals seems to be unique to this bacterium.
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http://dx.doi.org/10.1111/j.1758-2229.2010.00157.xDOI Listing
June 2010

Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions.

Microbiology (Reading) 2010 Jan 24;156(Pt 1):49-59. Epub 2009 Sep 24.

Department of Pharmaceutical Biology, University of Groningen, 9713 AV Groningen, The Netherlands.

PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.
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http://dx.doi.org/10.1099/mic.0.030973-0DOI Listing
January 2010

Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa.

J Biotechnol 2009 Jul 27;142(3-4):250-8. Epub 2009 May 27.

Department of Pharmaceutical Biology, University Center of Pharmacy, Groningen University Institute for Drug Exploration (GUIDE), Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 degrees C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
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http://dx.doi.org/10.1016/j.jbiotec.2009.05.005DOI Listing
July 2009

Enhanced antitumor efficacy of a DR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model.

Clin Cancer Res 2009 Mar 10;15(6):2048-57. Epub 2009 Mar 10.

Department of Medical Oncology, University Medical Center Groningen, University of Groningen, The Netherlands.

Purpose: Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is clinically evaluated as novel anticancer drug. rhTRAIL-DR5, a rhTRAIL variant that specifically binds to DR5 receptor, has recently been developed. We investigated whether rhTRAIL-DR5 is more efficient than rhTRAIL in combination with cisplatin in DR5-expressing human A2780 ovarian cancer cells.

Design: Effect of cisplatin alone or in combination with rhTRAIL or rhTRAIL-DR5 on DR5 surface expression, apoptosis, and cell survival of A2780 was measured. Biodistribution analysis was done in mice with (125)I-rhTRAIL administered intravenously versus intraperitoneally. Antitumor efficacy of rhTRAIL-DR5 versus rhTRAIL was determined in an intraperitoneally growing bioluminescent A2780 xenograft model.

Results: Cisplatin strongly enhanced DR5 surface expression. Both rhTRAIL and rhTRAIL-DR5 in combination with cisplatin induced high levels of caspase-3 activation, apoptosis, and cell kill, with rhTRAIL-DR5 being most potent. Intraperitoneal administration of (125)I-rhTRAIL resulted in a 1.7-fold higher area under the curve in serum, increased tumor exposure, and more caspase-3 activation in the tumor than intravenous administration. Intraperitoneal administration of rhTRAIL-DR5 delayed A2780 tumor progression, reflected in a mean light reduction of 68.3% (P = 0.015), whereas rhTRAIL or rhTRAIL-DR5 plus cisplatin resulted in 85% (P = 0.003) and 97% (P = 0.002) reduction compared with A2780 tumor progression in vehicle-treated animals. Combination of rhTRAIL-DR5 with cisplatin was more effective than cisplatin alone (P = 0.027).

Conclusion: rhTRAIL-DR5 was superior over rhTRAIL in vitro and in vivo against DR5-expressing ovarian cancer also in combination with cisplatin. Intraperitoneal administration of rhTRAIL-DR5 warrants further exploration in ovarian cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-08-1535DOI Listing
March 2009

Enhancement of antitumor properties of rhTRAIL by affinity increase toward its death receptors.

Biochemistry 2009 Mar;48(10):2180-91

Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.
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http://dx.doi.org/10.1021/bi801927xDOI Listing
March 2009

DR4-selective tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) variants obtained by structure-based design.

J Biol Chem 2008 Jul 12;283(29):20560-8. Epub 2008 May 12.

Centre for Genomic Regulation, CRG-EMBL Systems Biology Unit, Barcelona, Spain.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. Different tumor types respond either to DR4 or to DR5 activation, and chemotherapeutic drugs can increase the expression of DR4 or DR5 in cancer cells. Thus, DR4 or DR5 receptor-specific TRAIL variants would permit new and tumor-selective therapies. Previous success in generating a DR5-selective TRAIL mutant using computer-assisted protein design prompted us to make a DR4-selective TRAIL variant. Technically, the design of DR4 receptor-selective TRAIL variants is considerably more challenging compared with DR5 receptor-selective variants, because of the lack of a crystal structure of the TRAIL-DR4 complex. A single amino acid substitution of Asp at residue position 218 of TRAIL to His or Tyr was predicted to have a favorable effect on DR4 binding specificity. Surface plasmon resonance-based receptor binding tests showed a lowered DR5 affinity in concert with increased DR4 specificity for the designed variants, D218H and D218Y. Binding to DcR1, DcR2, and osteoprotegerin was also decreased. Cell line assays confirmed that the variants could not induce apoptosis in DR5-responsive Jurkat and A2780 cells but were able to induce apoptosis in DR4-responsive EM-2 and ML-1 cells.
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http://dx.doi.org/10.1074/jbc.M800457200DOI Listing
July 2008

A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A.

Chembiochem 2008 May;9(7):1110-5

Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands.

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.
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http://dx.doi.org/10.1002/cbic.200700754DOI Listing
May 2008

Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system.

Appl Environ Microbiol 2008 Mar 11;74(5):1402-11. Epub 2008 Jan 11.

Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.
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http://dx.doi.org/10.1128/AEM.01632-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258621PMC
March 2008

A highly active adipyl-cephalosporin acylase obtained via rational randomization.

FEBS J 2007 Nov 8;274(21):5600-10. Epub 2007 Oct 8.

Pharmaceutical Biology, University Centre for Pharmacy, University of Groningen, The Netherlands.

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl-7-aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36-fold. The activity of this mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.
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http://dx.doi.org/10.1111/j.1742-4658.2007.06081.xDOI Listing
November 2007
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