Publications by authors named "Laura Job"

7 Publications

  • Page 1 of 1

Inhibition of an organophosphate-detoxifying bacterial phosphotriesterase by albumin and plasma thiol components.

Toxicol Lett 2021 Oct 22;350:194-201. Epub 2021 Jul 22.

Chair of Biological Chemistry, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Electronic address:

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.
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http://dx.doi.org/10.1016/j.toxlet.2021.07.011DOI Listing
October 2021

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro.

Arch Toxicol 2021 08 23;95(8):2815-2823. Epub 2021 Jun 23.

Institut für Pharmakologie und Toxikologie der Bundeswehr, 80937, Munich, Germany.

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured k/K values were as high as 6.5 and 1.5 × 10 M min with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.
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http://dx.doi.org/10.1007/s00204-021-03094-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298220PMC
August 2021

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition.

Biochemistry 2020 11 4;59(45):4395-4406. Epub 2020 Nov 4.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from and , whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs , and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.
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http://dx.doi.org/10.1021/acs.biochem.0c00751DOI Listing
November 2020

Breast cancer survival in Brazil: How much health care access impact on cancer outcomes?

Breast 2020 Dec 16;54:155-159. Epub 2020 Oct 16.

Breast Cancer Center, Hospital Moinhos de Vento, Porto Alegre, RS, Brazil; Post-Graduation Program in Pathology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil; Oncology Center, Hospital Moinhos de Vento, Porto Alegre, RS, Brazil.

Background: Breast cancer has a high incidence and increasing mortality in Southern Brazil. The present study evaluated clinical and sociodemographic characteristics, and their association with overall survival in a private cancer center.

Methods: 1113 breast cancer patients were included in this study. The association between survival and clinicopathological and sociodemographic characteristics was analyzed using Cox regression and Kaplan-Meyer curves.

Results: Median age at diagnosis was 52 years (SD 13.5). Most patients were diagnosed in stages 0 and I (62.7%), while only 1.3% had stage IV disease. Five- and 10-year overall survival were 93.5% and 83.8%, respectively. According to multivariate analysis, age at diagnosis (HR 1.05; CI95 1.03-1.06), staging (stage III: HR 4.04; CI95 1.34-12.19; stage IV: HR 9.61; CI95 2.17-42.50), high KI67 (HR 5.46; CI95 1.27-23.32) and distant recurrence (HR 7.28; CI95 4.79-11.06) were significantly associated with survival. Smoking status, years of education, BMI, and tumor biological status were not significantly associated with mortality.

Conclusions: This cohort of Brazilian patients, who received timely and appropriate treatment, achieved outcomes that are comparable to those from high income countries. Breast cancer mortality seems dependent on the quality of health care available to patients.
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http://dx.doi.org/10.1016/j.breast.2020.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586236PMC
December 2020

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Toxicol Lett 2020 Mar 28;321:138-145. Epub 2019 Dec 28.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Electronic address:

Organophosphorus (OP) nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE), engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.030DOI Listing
March 2020

Time-course and degradation rate of membrane scaffold protein (MSP1D1) during recombinant production.

Biotechnol Rep (Amst) 2018 Mar 15;17:45-48. Epub 2017 Dec 15.

Institute of Food Science and Biotechnology (150), Department of Bioprocess Engineering (150k), University of Hohenheim, Fruwirthstraße 12, 70599, Stuttgart, Germany.

Membrane scaffold proteins (MSPs) are synthetic derivatives of apolipoprotein A-I, a major protein component of human high-density lipoprotein complexes. The most common among these is the variant MSP1D1, which has been in the focus of research on membrane mimetics in the past. As such, the amphipathic MSP1D1 has the ability to self-assemble in the presence of synthetic phospholipids into discoidal nanoparticles, so called nanodiscs. The recombinant production of MSP is exclusively reported using a standard laboratory expression system of the pET family. However, strong variations in both yield and achieved concentration as well as complications related to unspecific degradation are commonly reported. In addition, the time-course of recombinant protein as well as specific protein yields have not yet been quantified conclusively. In this study, the time-course of MSP1D1 concentration was investigated in a standard pET expression system in terms of quantification of production and degradation rates in comparison to a reference protein (eGFP).
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http://dx.doi.org/10.1016/j.btre.2017.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773449PMC
March 2018

New system to predict chemotherapeutic efficacy of drug combinations in fresh tumor samples.

PeerJ 2017 2;5:e3030. Epub 2017 Mar 2.

University Hospital for Gynecology, Carl von Ossietzky University Oldenburg, Germany.

Background: To find the best individual chemotherapy for cancer patients, the efficacy of different chemotherapeutic drugs can be predicted by pretesting tumor samples via the chemotherapy-resistance (CTR)-Test. Although drug combinations are widely used among cancer therapy, so far only single drugs are tested by this and other tests. However, several first line chemotherapies are combining two or more chemotherapeutics, leading to the necessity of drug combination testing methods.

Methods: We established a system to measure and predict the efficacy of chemotherapeutic drug combinations with the help of the Loewe additivity concept in combination with the CTR-test. A combination is measured by using half of the monotherapy's concentration of both drugs simultaneously. With this method, the efficacy of a combination can also be calculated based on single drug measurements.

Results: The established system was tested on a data set of ovarian carcinoma samples using the combination carboplatin and paclitaxel and confirmed by using other tumor species and chemotherapeutics. Comparing the measured and the calculated values of the combination testings revealed a high correlation. Additionally, in 70% of the cases the measured and the calculated values lead to the same chemotherapeutic resistance category of the tumor.

Conclusion: Our data suggest that the best drug combination consists of the most efficient single drugs and the worst drug combination of the least efficient single drugs. Our results showed that single measurements are sufficient to predict combinations in specific cases but there are exceptions in which it is necessary to measure combinations, which is possible with the presented system.
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http://dx.doi.org/10.7717/peerj.3030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337084PMC
March 2017
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