Çàïðîñ:quax wj[Author]
       Limits Activated: published in the last 3 years 
Results:23
1. FEBS J. 2010 Feb 12. [Epub ahead of print]

Synthetic constrained peptide selectively binds and antagonizes death receptor 5.

Vrielink J, Heins MS, Setroikromo R, Szegezdi E, Mullally MM, Samali A, Quax WJ.

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

Apoptosis or programmed cell death is an inherent part of the development and
homeostasis of multicellular organisms. Dysregulation of apoptosis is implicated 
in the pathogenesis of diseases such as cancer, neurodegenerative diseases and
autoimmune disorders. Tumour necrosis factor-related apoptosis-inducing ligand
(TRAIL) is able to induce apoptosis by binding death receptor (DR)4 (TRAIL-R1)
and DR5 (TRAIL-R2), which makes TRAIL an interesting and promising therapeutic
target. To identify peptides that specifically interact with DR5, a
disulfide-constrained phage display peptide library was screened for binders
towards this receptor. Phage-displayed peptides were identified that bind
specifically to DR5 and not to DR4, nor any of the decoy receptors. We show that 
the synthesized peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds 
specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited. Surface
plasmon resonance studies showed higher affinity towards DR5 for the dimeric form
then the monomeric form of the peptide, with apparent K(d) values of 40 nm versus
272 nm, respectively. Binding studied on cell lines by flow cytometry analyses
showed concentration-dependent binding. Upon co-incubation with increasing
concentrations of TRAIL, the peptide binding was reduced. Moreover, both the
monomeric and dimeric forms of the peptide reduced TRAIL-induced cell death in
Colo205 colon carcinoma cells. The peptide, YCKVILTHRCY, or its derivates, may be
a useful investigative tool for dissecting signalling via DR5 relative to DR4 or 
could act as a lead peptide for the development of therapeutic agents in diseases
with dysregulated TRAIL-signalling.

PMID: 20156289 [PubMed - as supplied by publisher]


2. Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):686-91. Epub 2009 Dec 22.

The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase
with an unusual substrate-binding pocket.

Bokhove M, Jimenez PN, Quax WJ, Dijkstra BW.

Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 
AG Groningen, The Netherlands.

In many Gram-negative pathogens, their virulent behavior is regulated by quorum
sensing, in which diffusible signals such as N-acyl homoserine lactones (AHLs)
act as chemical messaging compounds. Enzymatic degradation of these diffusible
signals by, e.g., lactonases or amidohydrolases abolishes AHL regulated
virulence, a process known as quorum quenching. Here we report the first crystal 
structure of an AHL amidohydrolase, the AHL acylase PvdQ from Pseudomonas
aeruginosa. PvdQ has a typical alpha/beta heterodimeric Ntn-hydrolase fold,
similar to penicillin G acylase and cephalosporin acylase. However, it has a
distinct, unusually large, hydrophobic binding pocket, ideally suited to
recognize C12 fatty acid-like chains of AHLs. Binding of a C12 fatty acid or a
3-oxo-C12 fatty acid induces subtle conformational changes to accommodate the
aliphatic chain. Furthermore, the structure of a covalent ester intermediate
identifies Serbeta1 as the nucleophile and Asnbeta269 and Valbeta70 as the
oxyanion hole residues in the AHL degradation process. Our structures show the
versatility of the Ntn-hydrolase scaffold and can serve as a structural paradigm 
for Ntn-hydrolases with similar substrate preference. Finally, the
quorum-quenching capabilities of PvdQ may be utilized to suppress the
quorum-sensing machinery of pathogens.

PMCID: PMC2818923 [Available on 2010/7/12]
PMID: 20080736 [PubMed - indexed for MEDLINE]


3. Appl Environ Microbiol. 2010 Feb;76(3):659-69. Epub 2009 Nov 30.

Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to
secretion of a heterologous protein by Bacillus subtilis.

Kouwen TR, Nielsen AK, Denham EL, Dubois JY, Dorenbos R, Rasmussen MD, Quax WJ,
Freudl R, van Dijl JM.

Department of Medical Microbiology, University Medical Center Groningen, 9700 RB 
Groningen, The Netherlands.

Bacillus subtilis is a well-established cell factory for efficient secretion of
many biotechnologically relevant enzymes that are naturally produced by it or
related organisms. However, the use of B. subtilis as a host for production of
heterologous secretory proteins can be complicated by problems related to
inefficient translocation of the foreign proteins across the plasma membrane or
to inefficient release of the exported proteins from the cell surface into the
surrounding medium. Therefore, there is a clear need for tools that allow more
efficient membrane targeting, translocation, and release during the production of
these proteins. In the present study, we investigated the contributions of the
pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to 
secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia
coli, by B. subtilis. The results indicate that the presence of the
pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)),
contributes significantly to the efficient secretion of PhoA by B. subtilis and
that pre(lip) directs PhoA secretion more efficiently than the authentic signal
peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses 
indicate that, under the conditions tested, no known secretion or membrane-cell
wall stress responses were provoked by the production of PhoA with any of the
pre- and pro-region sequences used. Our data underscore the view that the pre-pro
signals of the S. hyicus lipase are very useful tools for secretion of
heterologous proteins in B. subtilis.

PMCID: PMC2812991 [Available on 2010/8/1]
PMID: 19948853 [PubMed - in process]


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

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

Nadal Jimenez P, Koch G, Papaioannou E, Wahjudi M, Krzeslak J, Coenye T, Cool RH,
Quax WJ.

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.

PMID: 19778968 [PubMed - indexed for MEDLINE]


5. Antimicrob Agents Chemother. 2009 Nov;53(11):4891-7. Epub 2009 Aug 31.

Quorum-quenching acylase reduces the virulence of Pseudomonas aeruginosa in a
Caenorhabditis elegans infection model.

Papaioannou E, Wahjudi M, Nadal-Jimenez P, Koch G, Setroikromo R, Quax WJ.

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

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone
(AHL) acylase capable of degrading N-(3-oxododecanoyl)-L-homoserine lactone by
cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in 
vitro in a number of phenotypic assays. To address the question of whether PvdQ
also shows quorum-quenching properties in vivo, an infection model based on the
nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay,
strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than
the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ)
survived and continued to grow when using this strain as a food source.
Interestingly, in a slow-killing assay monitoring the survival of the nematodes
throughout a 4-day course, strain PAO1-Delta pvdQ was shown to be more virulent
than the wild-type strain, confirming the role of PvdQ as a virulence-reducing
agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much
more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was
added to C. elegans worms infected with wild-type PAO1, and this resulted in
reduced pathogenicity and increased the life span of the nematodes. From our
observations we can conclude that PvdQ might be a strong candidate for
antibacterial therapy against Pseudomonas infections.

PMCID: PMC2772301 [Available on 2010/5/1]
PMID: 19721066 [PubMed - indexed for MEDLINE]


6. Chembiochem. 2009 Sep 4;10(13):2236-45.

Alteration of the diastereoselectivity of 3-methylaspartate ammonia lyase by
using structure-based mutagenesis.

Raj H, Weiner B, Veetil VP, Reis CR, Quax WJ, Janssen DB, Feringa BL, Poelarends 
GJ.

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

3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of
mesaconate to give both (2S,3S)-3-methylaspartic acid and
(2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is
likely to involve general base catalysis, in which a catalytic base abstracts the
C3 proton of the respective stereoisomer to generate an enolate anion
intermediate that is stabilized by coordination to the essential active-site
Mg(II) ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic
acid suggests that Lys331 is the only candidate in the vicinity that can function
as a general base catalyst. The structure of the complex further suggests that
two other residues, His194 and Gln329, are responsible for binding the C4
carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely
candidates to assist the Mg(II) ion in stabilizing the enolate anion
intermediate. In this study, the importance of Lys331, His194, and Gln329 for the
activity and stereoselectivity of MAL was investigated by site-directed
mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, 
whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or
histidine. The properties of the mutant proteins were investigated by circular
dichroism (CD) spectroscopy, kinetic analysis, and (1)H NMR spectroscopy. The CD 
spectra of all mutants were comparable to that of wild-type MAL, and this
indicates that these mutations did not result in any major conformational
changes. Kinetic studies demonstrated that the mutations have a profound effect
on the values of k(cat) and k(cat)/K(M); this implicates Lys331, His194 and
Gln329 as mechanistically important. The (1)H NMR spectra of the amination and
deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A
showed that these mutants have strongly enhanced diastereoselectivities. In the
amination direction, they catalyze the conversion of mesaconate to yield only
(2S,3S)-3-methylaspartic acid, with no detectable formation of
(2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism 
in which Lys331, His194, and Gln329 are involved in positioning the substrate and
in formation and stabilization of the enolate anion intermediate.

PMID: 19670200 [PubMed - indexed for MEDLINE]


7. FEBS J. 2009 Jun;276(11):2994-3007. Epub 2009 Apr 16.

Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia
lyase from Bacillus sp. YM55-1.

Puthan Veetil V, Raj H, Quax WJ, Janssen DB, Poelarends GJ.

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

Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible
deamination of L-aspartate to yield fumarate and ammonia. In the proposed
mechanism for these enzymes, an active site base abstracts a proton from C3 of
L-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of
this intermediate eliminates ammonia and yields the product, fumarate. Although
two crystal structures of aspartases have been determined, details of the
catalytic mechanism have not yet been elucidated. In the present study, eight
active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and 
Asn326) were mutated in the structurally characterized aspartase (AspB) from
Bacillus sp. YM55-1. On the basis of a model of the complex in which L-aspartate 
was docked manually into the active site of AspB, the residues responsible for
binding the amino group of L-aspartate were predicted to be Thr101, Asn142 and
His188. This postulate is supported by the mutagenesis studies: mutations at
these positions resulted in mutant enzymes with reduced activity and significant 
increases in the K(m) for L-aspartate. Studies of the pH dependence of the
kinetic parameters of AspB revealed that a basic group with a pK(a) of
approximately 7 and an acidic group with a pK(a) of approximately 10 are
essential for catalysis. His188 does not play the typical role of active site
base or acid because the H188A mutant retained significant activity and displayed
an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of
Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these 
residues are most likely involved in substrate binding and in stabilizing the
enediolate intermediate. Mutagenesis studies corroborate the essential role of
Lys324 because all mutations at this position resulted in mutant enzymes that
were completely inactive. The substrate-binding model and kinetic analysis of
mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1
carboxylate group of the substrate. A catalytic mechanism for AspB is presented
that accounts for the observed properties of the mutant enzymes. Several features
of the mechanism that are also found in related enzymes are discussed in detail
and may help to define a common substrate binding mode for the lyases in the
aspartase/fumarase superfamily.

PMID: 19490103 [PubMed - indexed for MEDLINE]


8. J Biotechnol. 2009 Jul 15;142(3-4):250-8. Epub 2009 May 27.

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

Krzeslak J, Braun P, Voulhoux R, Cool RH, Quax WJ.

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.

PMID: 19481123 [PubMed - indexed for MEDLINE]


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

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

Duiker EW, de Vries EG, Mahalingam D, Meersma GJ, Boersma-van Ek W, Hollema H,
Lub-de Hooge MN, van Dam GM, Cool RH, Quax WJ, Samali A, van der Zee AG, de Jong 
S.

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.

PMID: 19276284 [PubMed - indexed for MEDLINE]


10. Biochemistry. 2009 Mar 17;48(10):2180-91.

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

Reis CR, van der Sloot AM, Szegezdi E, Natoni A, Tur V, Cool RH, Samali A,
Serrano L, Quax WJ.

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.

PMID: 19236007 [PubMed - indexed for MEDLINE]


11. Appl Environ Microbiol. 2008 Dec;74(24):7536-45. Epub 2008 Oct 24.

Modulation of thiol-disulfide oxidoreductases for increased production of
disulfide-bond-containing proteins in Bacillus subtilis.

Kouwen TR, Dubois JY, Freudl R, Quax WJ, van Dijl JM.

Department of Medical Microbiology, University Medical Center Groningen and
University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The
Netherlands.

Disulfide bonds are important for the correct folding, structural integrity, and 
activity of many biotechnologically relevant proteins. For synthesis and
subsequent secretion of these proteins in bacteria, such as the well-known "cell 
factory" Bacillus subtilis, it is often the correct formation of disulfide bonds 
that is the greatest bottleneck. Degradation of inefficiently or incorrectly
oxidized proteins and the requirement for costly and time-consuming reduction and
oxidation steps in the downstream processing of the proteins still are major
limitations for full exploitation of B. subtilis for biopharmaceutical
production. Therefore, the present study was aimed at developing a novel in vivo 
strategy for improved production of secreted disulfide-bond-containing proteins. 
Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; 
introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and
addition of redox-active compounds to the growth medium. As shown using the
disulfide-bond-containing molecule Escherichia coli PhoA as a model protein,
combined use of these three approaches resulted in secretion of amounts of active
PhoA that were approximately 3.5-fold larger than the amounts secreted by the
parental strain B. subtilis 168. Our findings indicate that Bacillus strains with
improved oxidizing properties can be engineered for biotechnological production
of heterologous high-value proteins containing disulfide bonds.

PMCID: PMC2607156
PMID: 18952880 [PubMed - indexed for MEDLINE]


12. Chem Biol. 2008 Aug 25;15(8):782-9.

Loop grafting of Bacillus subtilis lipase A: inversion of enantioselectivity.

Boersma YL, Pijning T, Bosma MS, van der Sloot AM, Godinho LF, Dröge MJ, Winter
RT, van Pouderoyen G, Dijkstra BW, Quax WJ.

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

Lipases are successfully applied in enantioselective biocatalysis. Most lipases
contain a lid domain controlling access to the active site, but Bacillus subtilis
Lipase A (LipA) is a notable exception: its active site is solvent exposed. To
improve the enantioselectivity of LipA in the kinetic resolution of
1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the
active-site entrance by longer loops originating from Fusarium solani cutinase
and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the
interaction surface for the substrate. The resulting loop hybrids showed
enantioselectivities inverted toward the desired enantiomer of IPG. The
acetylxylan esterase-derived variant showed an inversion in enantiomeric excess
(ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to
an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was
further improved by directed evolution to an ee of +57.4%.

PMID: 18721749 [PubMed - indexed for MEDLINE]


13. Proteomics. 2008 Jul;8(13):2704-13.

Genetic or chemical protease inhibition causes significant changes in the
Bacillus subtilis exoproteome.

Westers L, Westers H, Zanen G, Antelmann H, Hecker M, Noone D, Devine KM, van
Dijl JM, Quax WJ.

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

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals.
However, the susceptibility of heterologous proteins to degradation by
(extracellular) proteases is a major limitation for use of B. subtilis as a
protein cell factory. An increase in protein production levels has previously
been achieved by using either protease-deficient strains or addition of protease 
inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical 
inhibition of proteases have thus far not been compared in a systematic way. In
the present studies, we therefore compared the exoproteomes of cells in which
extracellular proteases were genetically or chemically inactivated. The results
show substantial differences in the relative abundance of various extracellular
proteins. Furthermore, a comparison of the effects of genetic and/or chemical
protease inhibition on the stress response triggered by (over) production of
secreted proteins showed that chemical protease inhibition provoked a genuine
secretion stress response. From a physiological point of view, this suggests that
the deletion of protease genes is a better way to prevent product degradation
than the use of protease inhibitors. Importantly however, studies with human
interleukin-3 show that chemical protease inhibition can result in improved
production of protease-sensitive secreted proteins even in mutant strains lacking
eight extracellular proteases.

PMID: 18546160 [PubMed - indexed for MEDLINE]


14. J Biol Chem. 2008 Jul 18;283(29):20560-8. Epub 2008 May 12.

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

Tur V, van der Sloot AM, Reis CR, Szegezdi E, Cool RH, Samali A, Serrano L, Quax 
WJ.

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.

PMID: 18474604 [PubMed - indexed for MEDLINE]


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

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

Boersma YL, Dröge MJ, van der Sloot AM, Pijning T, Cool RH, Dijkstra BW, Quax WJ.

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.

PMID: 18383241 [PubMed - indexed for MEDLINE]


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

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

Krzeslak J, Gerritse G, van Merkerk R, Cool RH, Quax WJ.

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.

PMCID: PMC2258621
PMID: 18192420 [PubMed - indexed for MEDLINE]


17. Apoptosis. 2008 Feb;13(2):225-35.

RGD-avidin-biotin pretargeting to alpha v beta 3 integrin enhances the
proapoptotic activity of TNF alpha related apoptosis inducing ligand (TRAIL).

Tarrus M, van der Sloot AM, Temming K, Lacombe M, Opdam F, Quax WJ, Molema G,
Poelstra K, Kok RJ.

Department of Pharmacokinetics and Drug Delivery, Groningen University Institute 
for Drug Exploration, Groningen, The Netherlands.

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a
powerful and selective inducer of tumor cell death. We hypothesize that TRAIL's
potential as anticancer agent can be enhanced further by promoting its
accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that
bind to angiogenic endothelial cells. We employed an avidin-biotin pretargeting
approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The
assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. 
Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex
onto endothelial cells enhanced the tumor cell killing activity. Endothelial
cells were not killed by TRAIL nor its derived complexes. Our approach can
facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may
enhance intratumoral accumulation. Furthermore, it offers a versatile technology 
for the complexation of targeting ligands with therapeutic recombinant proteins
and by this a novel way to enhance their specificity and activity.

PMCID: PMC2217618
PMID: 18071905 [PubMed - indexed for MEDLINE]


18. Eur J Med Chem. 2008 Jun;43(6):1171-9. Epub 2007 Sep 19.

Metabolic stereoselectivity of cytochrome P450 3A4 towards deoxypodophyllotoxin: 
In silico predictions and experimental validation.

Julsing MK, Vasilev NP, Schneidman-Duhovny D, Muntendam R, Woerdenbag HJ, Quax
WJ, Wolfson HJ, Ionkova I, Kayser O.

Department of Pharmaceutical Biology, Graduate School for Drug Exploration
(GUIDE), University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen,
The Netherlands.

Deoxypodophyllotoxin is stereoselectively converted into epipodophyllotoxin by
recombinant human cytochrome P450 3A4 (CYP3A4). Further kinetic analysis revealed
that the Michaelis-Menten K(m) and V(max) for hydroxylation of
deoxypodophyllotoxin by CYP3A4 at C7 position were 1.93 microM and 1.48
nmol/min/nmol, respectively. Deoxypodophyllotoxin was subjected to automated
docking analysis in order to get better knowledge of the interaction between the 
CYP3A4 enzyme and the substrate, using the PatchDock algorithm with distance
constraints. Automated docking showed that the beta-hydrogen atom at C7 position 
is in the most appropriate binding orientation at the site of oxidation. The
docking results are consistent with the experimental data for the bioconversion
of deoxypodophyllotoxin into epipodophyllotoxin by CYP3A4. In addition, the
effects of five lignans, deoxypodophyllotoxin, epipodophyllotoxin,
podophyllotoxin, demethylenedeoxypodophyllotoxin, and demethylenepodophyllotoxin,
on CYP3A4 were compared in order to investigate the influence of the
methylenedioxy group on the biotransformation process, to give insight into the
mode of metabolization and to explain inhibitory activity of lignans.

PMID: 17998150 [PubMed - indexed for MEDLINE]


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

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

Otten LG, Sio CF, Reis CR, Koch G, Cool RH, Quax WJ.

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.

PMID: 17922842 [PubMed - indexed for MEDLINE]


20. Proteins. 2008 Feb 1;70(2):333-43.

The conformation of the extracellular binding domain of Death Receptor 5 in the
presence and absence of the activating ligand TRAIL: a molecular dynamics study.

Wassenaar TA, Quax WJ, Mark AE.

Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Department of 
Biophysical Chemistry, University of Groningen, 9747AG Groningen, The
Netherlands.

The Death Receptor 5 (DR5), a member of tumor necrosis factor receptor (TNFR)
superfamily of receptors, triggers apoptosis (programmed cell death) when
stimulated by its tridentate ligand TRAIL. Until recently it was generally
assumed that the activation of DR5 resulted from the recruitment of three
independent receptor units, leading to the trimerization of intracellular
domains. However, there is mounting evidence to suggest that, in the absence of
ligand, such cytokine receptors primarily reside as preformed complexes. In this 
work, molecular dynamics simulations of the TRAIL-DR5 complex, the unbound
receptor trimer and individual receptor monomers are compared to gain insight in 
the mechanism of activation. The results suggest that, in the absence of TRAIL,
DR5 has a strong propensity to self-associate and that this is primarily mediated
through interactions of the membrane proximal domains. The association of the
free receptors leads to a loss of the threefold symmetry found within the
receptor-ligand complex. The simulations suggest that the primary role of TRAIL
is to induce threefold-symmetry within the DR5 complex and to constrain the
receptor to a specific conformation. The implications of this in terms of the
mechanism by which the receptor switches from an inactive to an active state are 
discussed. (c) 2007 Wiley-Liss, Inc.

PMID: 17671978 [PubMed - indexed for MEDLINE]


21. Mol Microbiol. 2007 May;64(4):984-99.

Thiol-disulphide oxidoreductase modules in the low-GC Gram-positive bacteria.

Kouwen TR, van der Goot A, Dorenbos R, Winter T, Antelmann H, Plaisier MC, Quax
WJ, van Dijl JM, Dubois JY.

Department of Medical Microbiology, University Medical Center Groningen and
University of Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, The
Netherlands.

Erratum in:
    Mol Microbiol. 2007 Jun;64(5):1417.

Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs)
is a universally conserved mechanism for stabilizing extracytoplasmic proteins.
In Escherichia coli, disulphide bond formation requires a concerted action of
distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function
in other bacteria has remained largely unexplored. Here we focus on TDORs of
low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and
BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues
DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus 
species respectively. To compare the function of these TDORs, DsbA was produced
in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to
sustain different TDOR-dependent processes, including heterologous secretion of
E. coli PhoA, competence development and bacteriocin (sublancin 168) production. 
The results show that DsbA can function in all three processes. While BdbD needs 
a quinone oxidoreductase for activity, DsbA activity appears to depend on
redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. 
subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can
functionally replace these quinone oxidoreductases in sublancin production. Taken
together, our unprecedented findings imply that TDOR systems of low-GC
Gram-positive bacteria have a modular composition.

PMID: 17501922 [PubMed - indexed for MEDLINE]


22. Appl Microbiol Biotechnol. 2007 Jul;75(6):1377-84. Epub 2007 Apr 26.

Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus
subtilis.

Julsing MK, Rijpkema M, Woerdenbag HJ, Quax WJ, Kayser O.

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

In comparison to other bacteria Bacillus subtilis emits the volatile compound
isoprene in high concentrations. Isoprene is the smallest representative of the
natural product group of terpenoids. A search in the genome of B. subtilis
resulted in a set of genes with yet unknown function, but putatively involved in 
the methylerythritol phosphate (MEP) pathway to isoprene. Further identification 
of these genes would give the possibility to engineer B. subtilis as a host cell 
for the production of terpenoids like the valuable plant-produced drugs
artemisinin and paclitaxel. Conditional knock-out strains of putative genes were 
analyzed for the amount of isoprene emitted. Differences in isoprene emission
were used to identify the function of the enzymes and of the corresponding
selected genes in the MEP pathway. We give proof on a biochemical level that
several of these selected genes from this species are involved in isoprene
biosynthesis. This opens the possibilities to investigate the physiological
function of isoprene emission and to increase the endogenous flux to the
terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for 
the heterologous production of more complex terpenoids in B. subtilis.

PMCID: PMC1914294
PMID: 17458547 [PubMed - indexed for MEDLINE]


23. FEBS J. 2007 May;274(9):2181-95.

Selection strategies for improved biocatalysts.

Boersma YL, Dröge MJ, Quax WJ.

Department of Pharmaceutical Biology, Groningen University Institute for Drug
Exploration, the Netherlands.

Enzymes have become an attractive alternative to conventional catalysts in
numerous industrial processes. However, their properties do not always meet the
criteria of the application of interest. Directed evolution is a powerful tool
for adopting the characteristics of an enzyme. However, selection of the evolved 
variants is a critical step, and therefore new strategies to enable selection of 
the desired enzymatic activity have been developed. This review focuses on these 
novel strategies for selecting enzymes from large libraries, in particular those 
that are used in the synthesis of pharmaceutical intermediates and
pharmaceuticals.

PMID: 17448143 [PubMed - indexed for MEDLINE]