Team:TU-Munich/Project/Bioaccumulation

From 2013.igem.org

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==BioAccumulation==
==BioAccumulation==
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[[File:TUM13_General_principle_of_accumulation_of_persistent_chemicals_along_the_food_chain.jpg|thumb|right|350px|Figure 1: General principle of accumulation of persistent chemicals along the food chain]]
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[[File:TUM13_General_principle_of_accumulation_of_persistent_chemicals_along_the_food_chain.jpg|thumb|right|350px|'''Figure 1:''' General principle of accumulation of persistent chemicals along the food chain.]]
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Bioaccumulation is a versatile and ambiguous concept to be defined. “General term describing a process by which chemicals are taken up by an organism either directly from exposure to a contaminated medium or by consumption of food containing the chemical.” is the definition of bioaccumulation of the [http://toxics.usgs.gov/definitions/bioaccumulation.html U.S. Environmental Protection Agency, 2010]. The majority of definitions is aimed at the vicious circle of the predator-prey relationship caused by the bioaccumulation which shows the build up of persistent chemicals such as DDT, PBC or dioxins in aquatic and terrestrial organisms. Health problems in humans, the survival of some affected species and overall biodiversity in aquatic and terrestrial ecosystems are at risk and a result of the consume and the accumulation of persistent chemicals along the food chain (see Figure 1). As most of these persistent chemicals are not biodegradable, an other approach is necessary.
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Bioaccumulation is a difficult concept to define. The [http://toxics.usgs.gov/definitions/bioaccumulation.html U.S. Environmental Protection Agency, 2010] defines bioaccumulation as a “general term describing a process by which chemicals are taken up by an organism either directly from exposure to a contaminated medium or by consumption of food containing the chemical”. The majority of definitions is aimed at the '''build up of persistent chemicals''' such as DDT, PBC or dioxins along the food chain (see figure 1), where the pollutants accumulate in predators until they become toxic. This can lead to the local and global extinction of species and health problems for humans. As most of these persistent chemicals are not biodegradable '''a different approach is necessary'''.
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[[File:TUM13 General_principle_bioaccumulation.png|thumb|left|350px|Figure 2: General principle of our bioaccumulation]]
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[[File:TUM13 General_principle_bioaccumulation.png|thumb|left|350px|'''Figure 2:''' Principle of our bioaccumulation system.]]
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Using our transgenic moos ([https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] ) as chassis, we can produce effectors for specific binding of pollutants. For the sustained removal of the target pollutant the localization of the effectors must be membrane associated in contrast to the cytoplasmatic or secretory localization of biodegrading effectors. The membrane association ensures the fixation of the pollutant to the membrane after successful binding. Internalisation of the effectors either results in metabolisation or increased targeted bioaccumulation of the pollutant in the transgenic plant cell. In case of bioaccumulation of the pollutant, the regular, safe disposal of parts of the plants contained in the filter would be necessary.  
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Using our transgenic moos ([https://2013.igem.org/Team:TU-Munich/Project/Physcomitrella ''Physcomitrella patens''] ) as a chassis, we can produce effectors for specific binding of pollutants. For the sustained removal of the target pollutant the effectors must be membrane-anchored, in contrast to the cytoplasmic or secretory localization of biodegrading effectors. The membrane association ensures the fixation of the pollutant to the membrane after successful binding. Internalisation of the effectors either results in metabolism or accumulation of the pollutant in the transgenic plant cell. In case of accumulation, controlled disposal of the plants in the filter would be necessary.  
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There is a broad range of natural as well as engineered binding proteins available. Natural binding proteins act as a model and initiator in design of new artificial binding proteins regarding research in different fields of biotechnology. Lipocalins, natural binding proteins, as base and scaffold for the design of anticalins confirms the popularity of bioaccumulated proteins in red biotechnology [[http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber et al., 2005]]. The role of Fibrillin in the abscisic acid-mediated photoprotection shows an example of functioned bioaccumulation in a plant  [[http://www.pnas.org/content/103/15/6061 Yang et al.,2007]]. The most commonly known binding proteins are antibodies which defend mammals against pathogens and toxins. Beside these natural binding proteins and anticalins  there are more and more designed binding proteins such as Affibodies derived from the z-domain of the antibody-binding protein A (Ref) and DARPins that are based on an ankyrin scaffold.One research topic of the [http://www.ws.chemie.tu-muenchen.de chair of analytical chemistry of the TU Munich] is the depletion of algal toxin contaminated water by using selective biofilters based on plantibodies. Plantibodies are plant- produced and derived antibodies which can construct antibody-mediated pathogen resistance as well as change the plant phenotype by immunomodulation [[http://www.ncbi.nlm.nih.gov/pubmed/11950570 Stoger et al.,2002]].
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There is a broad range of natural as well as engineered binding proteins available. Natural binding proteins act as a model and initiator in design of new artificial binding proteins combining research in different fields of biotechnology. Lipocalins, natural binding proteins, as base and scaffold for the design of anticalins confirm the popularity of bioaccumulated proteins in red biotechnology [[http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber et al., 2005]]. The role of Fibrillin in the abscisic acid-mediated photoprotection shows an example of functioned bioaccumulation in a plant  [[http://www.pnas.org/content/103/15/6061 Yang et al.,2007]]. The most commonly known binding proteins are antibodies which defend mammals against pathogens and toxins. Besides these natural binding proteins and anticalins  there are more and more designed binding proteins such as Affibodies derived from the z-domain of the antibody-binding protein A (Ref) and DARPins that are based on an ankyrin scaffold. One research topic of the [http://www.ws.chemie.tu-muenchen.de chair of analytical chemistry of the TU Munich] is the depletion of algal toxin contaminated water by using selective biofilters based on plantibodies. Plantibodies are plant- produced and derived antibodies which can construct antibody-mediated pathogen resistance as well as change the plant phenotype by immunomodulation [[http://www.ncbi.nlm.nih.gov/pubmed/11950570 Stoger et al.,2002]].
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For our project we exemplarily chose three different binding proteins which each exploit different mechanisms to bind Proteins.
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For our project we exemplarily choose three different binding proteins which each exploit different mechanisms to bind Proteins:
* The anticalin [[#Bioaccumulation_via_binding_proteins| <b>fluA</b>]] as an example for a binding protein.
* The anticalin [[#Bioaccumulation_via_binding_proteins| <b>fluA</b>]] as an example for a binding protein.
* The enzyme [[#Glutathione_S-transferase| <b>Gluthathione-S-Transferase</b>]] as an example where the formation of covalent bonds between the target pollutant and another molecule (gluthatione) is catalized.
* The enzyme [[#Glutathione_S-transferase| <b>Gluthathione-S-Transferase</b>]] as an example where the formation of covalent bonds between the target pollutant and another molecule (gluthatione) is catalized.
* The enzyme [[#Protein_Phosphotase_1_-_A_molecular_mop_for_Microcystin| <b>Protein Phosphatase 1</b>]] as example for a target of a binding protein. Here we exploit the inhibitory binding of the pollutant to the enzyme.
* The enzyme [[#Protein_Phosphotase_1_-_A_molecular_mop_for_Microcystin| <b>Protein Phosphatase 1</b>]] as example for a target of a binding protein. Here we exploit the inhibitory binding of the pollutant to the enzyme.
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To sketch the theoretical binding to a membrane anchored protein in our video, we created a short animation of the process using the molecularMaya software:
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<html>
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<center><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/77974681" width="450" height="255" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></center></html>
== Bioaccumulation via binding proteins ==
== Bioaccumulation via binding proteins ==
=== Problem Example: Diclofenac===
=== Problem Example: Diclofenac===
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Are pharmaceuticals a blessing or a curse? Respective to the ecological consequences of diclofenac this task must be valued negative. Diclofenac which is a proven, nonsteroidal anti-imflammatory drug (NSAID) is used mostly in the human medicine to treat a variety of acute and chronic pain and inflammatory conditions via inhibition of prostaglandin synthesis by inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)[[http://www.ncbi.nlm.nih.gov/pubmed/20470236 Gan,2010]]. Its necessity and importance in medicine research confirms e.g. the development of diclofenac patches for topical treatment of acute impact injuries [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1724805/ Predel et al.,2004]] and the main studies about safety and efficiency of the analgesic, anti-inflammatory, and antipyretic properties of diclofenac [[http://www.ncbi.nlm.nih.gov/pubmed/11276273 Morgan et al.,2001]][[http://www.ncbi.nlm.nih.gov/pubmed/15480981 Chan et al.,2004]][[http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CHsQFjAI&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F22168216&ei=DLI5UqrPNJHSsgaL2YHAAQ&usg=AFQjCNFs7U5eWUrOKgcIuLly-afQqXpwTQ&sig2=ebhoYAq_NdG9v2nuxdB8BQ&bvm=bv.52288139,d.Yms Pavelka,2012]]. The possible entered vicious circle (see figure 1) shows the poisoning tragedy in 2005 which leads to mass mortality and decline of vultures species especially ''Gyps bengalensis'', ''Gyps indicus'' and ''Gyps tenuirostris'' across the Indian sub-continent. Based on the consume of diclofenac by animal cadaver which are the main food source of vultures is the result of the eradication, breeding programs and reduction of the species diversity.
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Are pharmaceuticals a blessing or a curse? Respective to the ecological consequences of diclofenac this task must be valued negative. Diclofenac which is a proven, nonsteroidal anti-imflammatory drug (NSAID) is used mostly in human medicine to treat a variety of acute and chronic pain and inflammatory conditions via inhibition of prostaglandin synthesis by inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)[[http://www.ncbi.nlm.nih.gov/pubmed/20470236 Gan,2010]]. Its necessity and importance in medicine research confirm e.g. the development of diclofenac patches for topical treatment of acute impact injuries [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1724805/ Predel et al.,2004]] and the main studies about safety and efficiency of the analgesic, anti-inflammatory, and antipyretic properties of diclofenac [[http://www.ncbi.nlm.nih.gov/pubmed/11276273 Morgan et al.,2001]][[http://www.ncbi.nlm.nih.gov/pubmed/15480981 Chan et al.,2004]][[http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CHsQFjAI&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F22168216&ei=DLI5UqrPNJHSsgaL2YHAAQ&usg=AFQjCNFs7U5eWUrOKgcIuLly-afQqXpwTQ&sig2=ebhoYAq_NdG9v2nuxdB8BQ&bvm=bv.52288139,d.Yms Pavelka,2012]]. The possible resulting vicious circle (see Figure 1) shows the poisoning tragedy in 2005 which led to mass mortality and decline of vultures species especially ''Gyps bengalensis'', ''Gyps indicus'' and ''Gyps tenuirostris'' across the Indian sub-continent. Based on the consumation of diclofenac by animals, vultures have an abnormally high intake in this drug by eating the cadavers of contaminated animals.
=== Current solutions===
=== Current solutions===
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The fate of degradation products with their toxicity character and the development of antibiotic resistances plays a major role of the research on the effect of diclofenac in the aquatic environment. Because of the lack of analytical standards and the complex matrix of organic molecules in environmental samples, the degradation products have not yet been identified in molecular structure or concentration in aquatic fauna and flora.  
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The fate of degradation products with their toxicity character and the development of antibiotic resistances plays a major role of the research on the effect of diclofenac in the aquatic environment. Because of the lack of analytical standards and the complex matrix of organic molecules in environmental samples, the degradation products have not yet been identified regarding molecular structure or concentration in aquatic fauna and flora.  
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The degree of the problem becomes visible in the HPLC analysis, which shows in addition to the fast degradation of diclofenac over the irradiation time, a phytotoxicity increase of the increased degradation product concentrations and a weak inhibition of cell growth.
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The degree of the problem becomes visible in HPLC analysis, which shows in addition to the fast degradation of diclofenac over the irradiation time, a phytotoxicity increase of the increasing degradation product concentrations and a weak inhibition of cell growth.
To reduce the existence of PhACs such as diclofenac, one possibility is the usage of sewage treatment plants (STPs). Their removal efficiency depends on the sludge retention time and on the characteristics of the pharmaceutical molecule. Other strategies are the adding of an additional stage to the STP. Another approach is treated in the publications [[http://www.ncbi.nlm.nih.gov/pubmed/16190224 Westerhoff et al. (2005)]][[http://www.ncbi.nlm.nih.gov/pubmed/15984812 Huber et al., (2005)]][[http://www.ncbi.nlm.nih.gov/pubmed/12697241 Ternes et al., (2003)]] which confirm a high removal efficiency up to 95% for these additional stages. Currently developments are the nano-filtration, ozonation and power activated carbon. The importance of knowing the transformation products before applying a new technology is again demonstrated by the fact that ozonation reduces the parental substance diclofenac but cannot mineralize it.
To reduce the existence of PhACs such as diclofenac, one possibility is the usage of sewage treatment plants (STPs). Their removal efficiency depends on the sludge retention time and on the characteristics of the pharmaceutical molecule. Other strategies are the adding of an additional stage to the STP. Another approach is treated in the publications [[http://www.ncbi.nlm.nih.gov/pubmed/16190224 Westerhoff et al. (2005)]][[http://www.ncbi.nlm.nih.gov/pubmed/15984812 Huber et al., (2005)]][[http://www.ncbi.nlm.nih.gov/pubmed/12697241 Ternes et al., (2003)]] which confirm a high removal efficiency up to 95% for these additional stages. Currently developments are the nano-filtration, ozonation and power activated carbon. The importance of knowing the transformation products before applying a new technology is again demonstrated by the fact that ozonation reduces the parental substance diclofenac but cannot mineralize it.
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To sum it up, biodegradation of diclofenac introduces severals new problems as '''degradation products are toxic''' and pose new harms to the environment.
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To sum it up, biodegradation of diclofenac introduces several new problems as '''degradation products are toxic''' and pose new harms to the environment.
===Bioaccumulation instead of Biodegradation===  
===Bioaccumulation instead of Biodegradation===  
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As biodegradation does not conclusively solve the problem of diclofenac in the aquatic environment, we propose the utilisation of antibodies for accumulation of diclofenac, which would solve the problem of toxic degradation products.
As biodegradation does not conclusively solve the problem of diclofenac in the aquatic environment, we propose the utilisation of antibodies for accumulation of diclofenac, which would solve the problem of toxic degradation products.
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Although an antibody against diclofenac has been developed [[http://www.ncbi.nlm.nih.gov/pubmed/12966990 Deng et al. (2003)]], in vivo verification of successful binding would not be straightforward. Therefore we aimed for a first proof of principle and decided to investigate another substance that is targetable by antibodies for which verification is feasible. One attractive option is the well-known fluorophore fluorescein which was used by the [[https://2008.igem.org/Team:FreiburgiGEM team Freiburg 2008]]. Fluorescein fluoresces until bound by the binding protein fluA, which makes characterization tractable. The covalent binding of fluA and flourescein is shown in Figure 3.
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Although an antibody against diclofenac has been developed [[http://www.ncbi.nlm.nih.gov/pubmed/12966990 Deng et al. (2003)]], in vivo verification of successful binding would not be straightforward. Therefore we aimed for a first proof of principle and decided to investigate another substance that is targetable by antibodies for which verification is feasible. One attractive option is the well-known fluorophore fluorescein which was used by the [https://2008.igem.org/Team:FreiburgiGEM team Freiburg 2008]. Fluorescein fluoresces until bound by the binding protein fluA, which makes characterization tractable. The covalent binding of fluA and flourescein is shown in Figure 3.
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We submitted an improved version of the Freiburg Biobrick for fluA (see Table 1) and created a transgenic moss plants ([[https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-15]]) with the fluorescein binding anticalin fluA on the extracellular part of the receptor.  
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We submitted an improved version of the Freiburg Biobrick for fluA (see Table 1) and created a transgenic moss plants ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-15]) with the fluorescein binding anticalin fluA on the extracellular part of the receptor.  
Producing engineered binding proteins for medical purpose in the red biotechnology is one of the main research fields in which anticalins play a major role. Lipocalins such as bilin-binding-protein BBP (from ''Pieris brassicae'') can be used for generating molecular pockets with a diversity of shapes and for creating a stable receptor protein for a ligand of choice, so that development of binding proteins against nearly chemical structures with comparable size is possible. Hence it should be possible to target a great variety of pollutants by already available binding proteins.
Producing engineered binding proteins for medical purpose in the red biotechnology is one of the main research fields in which anticalins play a major role. Lipocalins such as bilin-binding-protein BBP (from ''Pieris brassicae'') can be used for generating molecular pockets with a diversity of shapes and for creating a stable receptor protein for a ligand of choice, so that development of binding proteins against nearly chemical structures with comparable size is possible. Hence it should be possible to target a great variety of pollutants by already available binding proteins.
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|-
|-
|FluA
|FluA
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|152 nM
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| align=right | 152 nM
|[[http://www.ncbi.nlm.nih.gov/pubmed/10051566 Beste et al., 1999]]
|[[http://www.ncbi.nlm.nih.gov/pubmed/10051566 Beste et al., 1999]]
|<partinfo>BBa_K157004</partinfo>
|<partinfo>BBa_K157004</partinfo>
|-
|-
|FluA (R95K)
|FluA (R95K)
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|64 nM
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| align=right | 64 nM
|[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]
|[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]
|not available as BioBrick
|not available as BioBrick
|-
|-
|FluA (R95K, A45I, S114T)
|FluA (R95K, A45I, S114T)
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|2 nM
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| align=right | 2 nM
|[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]
|[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]]
|<partinfo>BBa_K1159002</partinfo>
|<partinfo>BBa_K1159002</partinfo>
|}
|}
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==Glutathione S-transferase ==
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==Glutathione-S-transferase ==
=== Problem:  Dichlorodiphenyltrichloroethane (DDT)===
=== Problem:  Dichlorodiphenyltrichloroethane (DDT)===
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=== Caltech 2011 Solution: Covalent binding to Gluthathion via Gluthathione-S-Transferase ===
=== Caltech 2011 Solution: Covalent binding to Gluthathion via Gluthathione-S-Transferase ===
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[[File:TUM13_conjugation_of_GSH_and_a_xenobiotic.png|thumb|right|350px| '''Figure 4:''' Conjugation of GSH and a xenobiotic]]
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[[File:TUM13_conjugation_of_GSH_and_a_xenobiotic.png|thumb|right|300px| '''Figure 4:''' Conjugation of GSH and a xenobiotic]]
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In context to prove the bioaccumulation and to develop an effective methode of removing DDT, we envision the usage of the cytoplasmatic GST 1-1, also known as DDT Dehyrochlorinase. Along with the creation of transgenic ''Physcomitrella patens'' plants ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-13] ) we used the [https://2013.igem.org/Team:TU-Munich/Results/BioBricks biobrick] from the [https://2011.igem.org/Team:Caltech Caltech iGEM team 2011]. This team engineered bacteria which can degrade endocrine-disrupting chemicals such as DDT, synthetic estrogen in bodies of water to less toxic forms. Glutathion S-transferases (GSTs), an eukaryotic and prokaryotic phase II metabolic isozymes-family, catalyze the conjugation of reduced form of gluthatione (GSH, nucleophil) and xenobiotics (electrophil) to gluthatione-S-Conjugate via nucleophilic attack (see Figure 4).  
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In context to prove the bioaccumulation and to develop an effective methode of removing DDT, we envision the usage of the cytoplasmatic GST 1-1, also known as DDT Dehyrochlorinase. Along with the creation of transgenic ''Physcomitrella patens'' plants ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-13]) we used the [https://2013.igem.org/Team:TU-Munich/Results/BioBricks biobrick] from the [https://2011.igem.org/Team:Caltech Caltech iGEM team 2011]. This team engineered bacteria which can degrade endocrine-disrupting chemicals such as DDT, synthetic estrogen in bodies of water to less toxic forms. Glutathion S-transferases (GSTs), an eukaryotic and prokaryotic phase II metabolic isozymes-family, catalyze the conjugation of reduced form of gluthatione (GSH, nucleophil) and xenobiotics (electrophil) to gluthatione-S-Conjugate via nucleophilic attack (see Figure 4).  
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[[File:TUM13_equilibrium reaction of Gluthation.png|thumb|right|350px| '''Figure 5:''' Equilibrium reaction of gluthation]]
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[[File:TUM13_equilibrium reaction of Gluthation.png|thumb|right|300px| '''Figure 5:''' Equilibrium reaction of gluthation]]
The consequence is an increased solubility of the conjugates which leads to the removal of xenobiotics in form of conjugates via vacuole enclosure. The action of the specific transporters and the steady supply of GSH in the equilibrium reaction (see Figure 5) are the limited factors of the detoxification reaction. Along with the detoxification and cell signaling function GST’s act as transport proteins, which gave GST the previous name ligandin. Table 2 shows the 3 different superfamilies with their characteristics.  
The consequence is an increased solubility of the conjugates which leads to the removal of xenobiotics in form of conjugates via vacuole enclosure. The action of the specific transporters and the steady supply of GSH in the equilibrium reaction (see Figure 5) are the limited factors of the detoxification reaction. Along with the detoxification and cell signaling function GST’s act as transport proteins, which gave GST the previous name ligandin. Table 2 shows the 3 different superfamilies with their characteristics.  
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[[File:TUM13 Structure3D_of_GST.gif|thumb|right|300px| '''Figure 6:''' Molecular structure of Gluthation S-Transferase]]
{|cellspacing="0" border="1"
{|cellspacing="0" border="1"
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|Cytosolic proteins
|Cytosolic proteins
|alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega
|alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega
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|>40
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| align=right | >40
|-
|-
|Mitochondrial proteins
|Mitochondrial proteins
|kappa
|kappa
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|<25
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| align=right | <25
|-
|-
|microsomal (MAPEG= membrane-associated proteins in eicosanoid and glutathione metabolism) proteins
|microsomal (MAPEG= membrane-associated proteins in eicosanoid and glutathione metabolism) proteins
|subgroups I-IV
|subgroups I-IV
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|<25
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| align=right | <25
|}
|}
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Generally the three superfamilies differ mostly in structure  and sequence as only the cytosolic and the mitochondrial superfamily have a thiorexin like domain in which the glutathione binding site (G-site) is located [[http://www.ncbi.nlm.nih.gov/pubmed/21428697 Oakley A.,2011]]. The helix alpha 2 is the most variable secondary structure. Y-GST is the subgroup which activates glutathione via using tyrosine residues. S/C-GST uses serine/cysteine residues. GST binds the substrate at the hydrophobic H-site of the enzyme and GSH at the hydrophilic G-site which together form the active site of the enzyme(see figure 6).
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[[File:TUM13_Reaction_of_monochlorobimane_with_GSH.png|thumb|right|300px| '''Figure 7:''' Reaction of monochlorobimane with GSH ]]
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In research techniques GST will be used as so called GST-tags for separation, elucidation of direct protein-protein interaction and purification of the GST-fusion protein mostly by pull-down assay. So targeting GST with molecule therapeutics represents GTS as an attractive target for drug discovery [[http://www.ncbi.nlm.nih.gov/pubmed/16550164 McIlwain et al.,2006]]. A mammalian variant of GST, GSTP, plays a major role in cancer- development and potential drug/chemotherapeutic resistance in a majority of tumor cell lines: The inhibition of the pro-apoptotic pathway (JNK pathway) and the overexpression of GSTP in tumor cells lead to escape of apoptosis of the tumor cells mediated by non GSTP- substance-drugs [[http://www.ncbi.nlm.nih.gov/pubmed/8770536 Hayes et al.,2005]] [[http://www.ncbi.nlm.nih.gov/pubmed/20981235 Josephy,2010]] [[http://www.ncbi.nlm.nih.gov/pubmed/10971201 Hayes et al.,2000]] [[http://www.ncbi.nlm.nih.gov/pubmed/12563680 Fraser et al.,2003]].
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To avoid the time- and labor-intensive method HPLC following derivatization with 2-nitrobenzoic acid we used the common sensitive technique with monochlorobimane to measure GSH as a proof of principle. The adding of monochlorobimane to the culture medium leads to the conjugation of GSH to monochlorobimane catalyzed by DDT Dehyrochlorinase(see figure 7). The GSH-monochlorobimane conjugate can be measured fluorometrically [[http://www.ncbi.nlm.nih.gov/pubmed/11038270 Kamencic et al.,2000]].
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<div class="box-left">
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Generally the three superfamilies differ mostly in structure  and sequence as only the cytosolic and the mitochondrial superfamily have a thiorexin like domain in which the glutathione binding site (G-site) is located [[http://www.ncbi.nlm.nih.gov/pubmed/21428697 Oakley A.,2011]]. The helix alpha 2 is the most variable secondary structure. Y-GST is the subgroup which activates glutathione via using tyrosine residues. S/C-GST uses serine/cysteine residues. GST binds the substrate at the hydrophobic H-site of the enzyme and GSH at the hydrophilic G-site which together form the active site of the enzyme (see Figure 6).
 +
In research techniques GST is used as so called GST-tags for separation, elucidation of direct protein-protein interaction and purification of the GST-fusion protein mostly by pull-down assay. So targeting GST with molecule therapeutics represents GTS as an attractive target for drug discovery [[http://www.ncbi.nlm.nih.gov/pubmed/16550164 McIlwain et al.,2006]].
 +
A mammalian variant of GST, GSTP, plays a major role in cancer- development and potential drug/chemotherapeutic resistance in a majority of tumor cell lines: The inhibition of the pro-apoptotic pathway (JNK pathway) and the overexpression of GSTP in tumor cells lead to escape of apoptosis of the tumor cells mediated by non GSTP- substance-drugs [[http://www.ncbi.nlm.nih.gov/pubmed/8770536 Hayes et al.,1995]] [[http://www.ncbi.nlm.nih.gov/pubmed/20981235 Josephy,2010]] [[http://www.ncbi.nlm.nih.gov/pubmed/10971201 Hayes et al.,2000]] [[http://www.ncbi.nlm.nih.gov/pubmed/12563680 Fraser et al.,2003]].
 +
To avoid the time- and labor-intensive method HPLC following derivatization with 2-nitrobenzoic acid we used the common sensitive technique with monochlorobimane to measure GSH as a proof of principle. The adding of monochlorobimane to the culture medium leads to the conjugation of GSH to monochlorobimane catalyzed by DDT Dehyrochlorinase (see Figure 7). The GSH-monochlorobimane conjugate can be measured fluorometrically [[http://www.ncbi.nlm.nih.gov/pubmed/11038270 Kamencic et al.,2000]].
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[[File:TUM13 Structure3D_of_GST.gif|thumb|center|400px| Figure 6:molecular structure of Gluthation S-transferase]]
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==Protein Phosphotase 1 &ndash; A molecular mop for Microcystin==
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</div>
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<div class="box-right">
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[[File:TUM13_Reaction_of_monochlorobimane_with_GSH.png|thumb|center|400px| Figure 7: Reaction of monochlorobimane with GSH ]]
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</div>
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==Protein Phosphotase 1 - A molecular mop for Microcystin==
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=== The Problem: Algal Blooms ===
=== The Problem: Algal Blooms ===
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Which phenomenon implicates the common name “Florida red tide”? This term is used in annually along Florida waters where species known as ''Karenia brevis'' leads to a red colored alga bloom along the Florida coast. Algal blooms are a worldwide environmental problem which confirms the appointment of 10.000 people for cleaning up the algal bloom in Beijing for the Olympic discipline “sailing regatta” in 2008. Depending on the causative bacteria algal can bloom at an amount of concentrations of hundreds to thousands of cells per milliliter. An officially recognized threshold level isn’t public. Possible diverse factors which are the reason for the high affecting of bloom formation are listed followed:
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[[File:TUM13_Johannas_Stuff.png|thumb|center|900px| '''Figure 8:''' Eutrophication]]
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Which phenomenon implicates the common name “Florida red tide”? This term is used in annually along Florida waters where species known as ''Karenia brevis'' leads to a red colored alga bloom along the Florida coast. Algal blooms are a worldwide environmental problem! This becomes evident with the appointment of 10.000 people for cleaning up the algal bloom in Beijing for the Olympic discipline “sailing regatta” in 2008. Depending on the cause, bacteria algal can bloom at concentrations of hundreds to thousands of cells per milliliter. An officially recognized threshold level isn’t public. Possible diverse factors which are the reason for the high affecting of bloom formation are listed followed:
*Light intensity: cyanobacteria which form surface blooms have a higher  tolerance concerning high light intensities. This leads to lower growth rate than other phytoplankton organisms.
*Light intensity: cyanobacteria which form surface blooms have a higher  tolerance concerning high light intensities. This leads to lower growth rate than other phytoplankton organisms.
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*Gas vacuoles: Gas vesicles give the cyanobacteria a lower density than water
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*Gas vacuoles: Gas vesicles give the cyanobacteria a lower density than water.
*Growth rate: Slow growth rates require long water retention times to enable a bloom.
*Growth rate: Slow growth rates require long water retention times to enable a bloom.
*Phosphorous and nitrogen: A low ratio of these substance may favour a development of cyanobacteria blooms.
*Phosphorous and nitrogen: A low ratio of these substance may favour a development of cyanobacteria blooms.
*Temperature: maximum growth rates are attained by the most cyanobacteria above 25°C. Thus much bloom formation is during the summer.
*Temperature: maximum growth rates are attained by the most cyanobacteria above 25°C. Thus much bloom formation is during the summer.
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A possible cascade which is caused by the release of nutrients in the aquatic ecosystems shows figure 8.Another result confirms the spread and the persistence of cholera through algal blooms [[http://www.ncbi.nlm.nih.gov/pubmed/8155853 Epstein PR.,1993]]. Currently the so called harmful algal blooms are an additive for the detection of bacteria spread and their caused health effects to fauna and flora [[http://www.ncbi.nlm.nih.gov/pubmed/21135906 Ferrante et al.,2010]] [[http://www.ncbi.nlm.nih.gov/pubmed/17298369 Barlaan EA et al.,2007]]. The reduction of the uptake of toxic methylmercury in freshwater food webs by algal blooms confirms a positive reverse of algal blooms [[http://www.ncbi.nlm.nih.gov/pubmed/11904388 Pickhardt PC. et al.,2002]].
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A possible cascade which is caused by the release of nutrients in the aquatic ecosystems is shown in Figure 8. Another result confirms the spread and the persistence of cholera through algal blooms [[http://www.ncbi.nlm.nih.gov/pubmed/8155853 Epstein PR.,1993]]. Currently the so called harmful algal blooms are an additive for the detection of bacteria spread and their caused health effects to fauna and flora [[http://www.ncbi.nlm.nih.gov/pubmed/21135906 Ferrante et al.,2010]] [[http://www.ncbi.nlm.nih.gov/pubmed/17298369 Barlaan EA et al.,2007]]. The reduction of the uptake of toxic methylmercury in freshwater food webs by algal blooms confirms a positive reverse of algal blooms [[http://www.ncbi.nlm.nih.gov/pubmed/11904388 Pickhardt PC. et al.,2002]].
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[[File:TUM13_Eutrophication.png|thumb|right|350px| Figure 8: Eutrophication]]
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=== Current Solution to combat Algal blooms ===
=== Current Solution to combat Algal blooms ===
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It is well known that cyanobacteria produce most Toxins such as microcystin under species-specific conditions which are most favorable for their growth. As a result most studies concerning this term were called into life. So for the species they found out that the toxin production increased under light optimum condition of the relative bacteria species and on low and high pH. The usage of zinc was necessary for the increase of growth and toxin production. Dependent on nitrogen fixing respectively non-nitrogen fixing species the nitrogen and phosphorous rate in media was unnecessary respectively necessary concerning to the toxin production. Evidences of the role of plasmid, multi-enzyme complexes and peptide synthetase gene involvement in toxin production were also confirmed: e.g. knockout experiments showed that peptide synthetase genes are responsible for microcysteine production (Dittman et al.2006).Major problems are still the lack of knowledge regarding to occurrence and toxicity of cyanobacterial products like the diversity of LPS structures which cause allergic as well as toxic human health problems, the extremely weak scientific basis and poorly developed procedures for valuation on health effects. At TDI (tolerable daily intake) is one of the values on which the toxicology studies aimed. Based on analytic and sampling problems or inadequate scientific data safe practice guidelines can be assisted for the reducing of exposure. The human exposure to the toxins is a major point to combat the human health risk. The WHO guideline values base upon a number of assumptions which change locally or nationally according to the present situation. To fill the lack of knowledge of parental variants the HPLC method is commonly used also for the determination of references. Current treatment processes used at water surface plants are coagulation, sand filtration, and clarification. The negative site of these methods is the not effective removing and destroying of cyanotoxins. To combat this problem treatment systems like carbon- and membrane filtration were developed. External and internal accumulation of toxins is a further research topic since the inhibition of Mustard seedling development by of microcystein-LR in aquatic root solution (Kos et al. 1995). Food crops which can accumulate diverse types of cyanobacteria toxins are one concern. For preventing management open channels of communication, public participation in risk assessment and policy development for example are also important. Just to mention one management action: Alert Levels Framework is useful to provide a graduated response to the begin and progress of cyanobacterial blooms.
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It is well known that cyanobacteria produce most Toxins such as microcystin under species-specific conditions which are most favorable for their growth. As a result most studies concerning this topic try to tackle the problem from this point of view. For the investigated species, they found out that the toxin production increased under light optimum condition of the relative bacteria species and on low and high pH. The usage of zinc was necessary for the increase of growth and toxin production. Dependent on nitrogen fixing respectively non-nitrogen fixing species the nitrogen and phosphorous rate in media was unnecessary respectively necessary concerning to the toxin production. Evidences of the role of plasmid, multi-enzyme complexes and peptide synthetase gene involvement in toxin production were also confirmed: e.g. knockout experiments showed that peptide synthetase genes are responsible for microcysteine production.
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=== Dundee 2013 Solution to combat Algal blooms===
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Major problems are still the lack of knowledge regarding to occurrence and toxicity of cyanobacterial products like the diversity of LPS structures which cause allergic as well as toxic human health problems, the extremely weak scientific basis and poorly developed procedures for valuation on health effects. The TDI (tolerable daily intake) is one of the key features on which the toxicology studies are aimed. Based on analytic and sampling problems or inadequate scientific data safe practice guidelines can be assisted for the reducing of exposure.  
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Due to the deleterious results from the toxin release of algal blooms many risk assesments were borned to live[[http://www.ncbi.nlm.nih.gov/pubmed/10499991 Schaeffer et al., 1999 ]]. The effects of microcystin for the aquatic and terrestrial ecosystem confirms its role in the modulation of proliferation, apoptosis and proliferation of spermatogenic cells in vivo [[http://www.ncbi.nlm.nih.gov/pubmed/24025782 Zhou et al.,2013]].Our aim is the bioaccumulation of microcystin to the natural binding protein PP1 in context to give an possible effective solution combating an alga bloom released toxin microcystin.An useable application of PP1 with regard to bioremediation implicates the iGEM project 2013 of [https://2013.igem.org/Team:Dundee team Dundee]. PP1 as a natural binding protein accumulates the toxin microcystin(toxin released by Microcystis aeruginosato) combat the releases harm to mammals. The interaction of microcystin in form of the heptapeptide structure with three distinct regions of the PP1c shows figure 9. After converting the human PP1, given by [https://2013.igem.org/Team:TU-Munich/Team/Collaborations iGEM Dundee], in RFC 25 and constructing some expression plasmids we produced transgenic ''phytomitrella patens'' ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-14]) with PP1 and a biobrick of PP1 receptor.
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The initiator of fixing phosphate residues to proteins is an enzyme family called kinases. The adversaries of the phosphorylation process are the protein phosphatases. In general you classify them in three families: phosphoserine and phosphothreonine residues were dephosporylated by PPM and PPP families whereas PTP dehosphorylate phosphotyrosine amino acids. The role of regulation of cellular processes represent PP1 which is an ubiquitous eukaryotic enzyme belonging to the protein serine/threonine phosphatase class. The enormous variety and multifunctionality of PP1 concerning to cellular functions through the interaction of its catalytic subunit (PP1c) is written down in the publication "Functional diversity of protein phosphatase-1, a cellular economizer and reset button" [[http://www.ncbi.nlm.nih.gov/pubmed/14715909 Ceulemans et al.,2004]]. Next to wide diversity of biological function such as the regulation of blood-glucose levels and glycogen metabolism PP1 plays a major relevance in disease research which aims in the red biotechnology to produce new drug designs. The role of PP1 concerning to the HIV-1-transcription [[http://www.ncbi.nlm.nih.gov/pubmed/17266553 Nekhai et al.,2007]] and Alzheimer disease brains [[http://www.ncbi.nlm.nih.gov/pubmed/8395566 Gong et al.,1993]] illustrate this. Basically changes in the levels, phosphorylation status and conformation of the PP1c which consists of diverse regulatory subunits and domains hydrophobic grooves on the surface mostly through a short conserved binding motif RVxF leads to targeting of substrates and inhibitors. Together with the conserved structure PP1c has the metalloenzyme characteristic of PP1: next to the potential substrate/inhibitor binding grooves with the C-Terminus at the end of the grooves a central β-α-β-α-β scaffold at the active site positions the two metal ions (Mn, Fe) which are coordinated by one asparagines, two aspartic acids and three histidines [[http://www.nature.com/nature/journal/v376/n6543/abs/376745a0.html Goldberg et al.,2002]].
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The human exposure to the toxins is a major point to combat the human health risk. The WHO guideline values base upon a number of assumptions which change locally or nationally according to the present situation. To fill the lack of knowledge of parental variants the HPLC method is commonly used also for the determination of references. Current treatment processes used at water surface plants are coagulation, sand filtration, and clarification.  
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[[File:TUM13_PP1.png|thumb|right|350px| Figure 9: interaction of PP1 and microcystin [[http://www.rcsb.org/pdb/explore/explore.do?structureId=2BDX source:PDB]]]]
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==References:==
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The negative site of these methods is the ineffective removal and destruction of cyanotoxins. To combat this problem treatment systems like carbon- and membrane filtration were developed. External and internal accumulation of toxins is another research topic since the inhibition of Mustard seedling development by of microcystein-LR in aquatic root solution.
-
<!-- Ab hier richtige Referenzen einfügen -->
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=== Dundee 2013 Solution to combat Algal blooms===
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[[http://www.ncbi.nlm.nih.gov/pubmed/12697241 Ternes et al., (2003)]] Ternes, T. A., Stu ̈ber, J., Herrmann, N., McDowell, D., Ried, A., Kampmann, M., and Teiser, B. (2003). Ozonation: a tool for removal of pharmaceuticals, contrast media and musk fragrances from wastewater? ''Water Res'', 37(8):1976–82.
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[[File:TUM13_PP1.png|thumb|right|350px| '''Figure 9:''' interaction of PP1 and microcystin [[http://www.rcsb.org/pdb/explore/explore.do?structureId=2BDX source: PDB]]]]
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[[http://www.ncbi.nlm.nih.gov/pubmed/15984812 Huber et al., (2005)]] Huber, M. M., G ̈obel, A., Joss, A., Hermann, N., L ̈offler, D., McArdell, C. S., Ried, A., Siegrist, H., Ternes, T. A., and von Gunten, U. (2005). Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: a pilot study. ''Environ Sci Technol'', 39(11):4290–9.
+
Due to the deleterious results from the toxin release of algal blooms many risk assesments were have been developed [[http://www.ncbi.nlm.nih.gov/pubmed/10499991 Schaeffer et al., 1999]]. The effects of microcystin for the aquatic and terrestrial ecosystem have been proven to include the modulation of proliferation, apoptosis and proliferation of spermatogenic cells in vivo [[http://www.ncbi.nlm.nih.gov/pubmed/24025782 Zhou et al.,2013]], which makes it an attractive target for our project.
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[[http://www.ncbi.nlm.nih.gov/pubmed/16190224 Westerhoff et al. (2005)]] Westerhoff, P., Yoon, Y., Snyder, S., and Wert, E. (2005). Fate of endocrine- disruptor, pharmaceutical, and personal care product chemicals during simulated drinking water treatment processes. ''Environ Sci Technol'', 39(17):6649–63.
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For the bioaccumulation of microcystin, it is possible to use the enzyme PP1. Mycrocystin can inhibit the function of PP1 by binding to it, this interaction of microcystin, in form of a heptapeptide structure, with three distinct regions of the PP1c is depicted in Figure 9. Hence PP1 can act as natural binding partner for mycrocystin.
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[[http://www.ncbi.nlm.nih.gov/pubmed/12966990 Deng et al. (2003)]] Deng, A., Himmelsbach, M., Zhu, Q.-Z., Frey, S., Sengl, M., Buchberger, W., Niessner, R., and Knopp, D. (2003). Residue analysis of the pharmaceutical diclofenac in different water types using elisa and gc-ms. ''Environ Sci Technol'', 37(15):3422–9.
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As the Dundee iGEM team 2013 [https://2013.igem.org/Team:Dundee Team Dundee] was already working on this subject we contacted them and they kindly provided us with the gene that encodes PP1. After converting the human PP1, to <nowiki>RFC</nowiki> 25 and constructing some expression plasmids we produced transgenic ''phytomitrella patens'' ([https://2013.igem.org/Team:TU-Munich/Results/GM-Moss PF-14]) with PP1 and a biobrick of PP1 receptor.
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[[http://www.ncbi.nlm.nih.gov/pubmed/10051566 Beste et al., 1999]] Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold. ''PNAS'', 96(5):1898-903.
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==References:==
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[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.
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<!-- Ab hier richtige Referenzen einfügen -->
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[[http://www.ncbi.nlm.nih.gov/pubmed/9255793 Nord et al., 1997]] Nord K, Gunneriusson E, Ringdahl J, Ståhl S, Uhlén M, Nygren PA. (1997) Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain. ''Nature Biotech''. 15(8):772-7.
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[[http://toxics.usgs.gov/definitions/bioaccumulation.html Definition of Bioaccumulation,U.S. Department of the Interior | U.S. Geological Survey,2013]]
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[[http://toxics.usgs.gov/definitions/bioaccumulation.html Definition of Bioaccumulation, U.S. Department of the Interior | U.S. Geological Survey,2013]] Definiton of Bioaccumulation
[[http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber and Skerra,2005]] Schlehuber S, Skerra A.(2005)Lipocalins in drug discovery: from natural ligand-binding proteins to "anticalins".''Drug Discov Today''10(1):23-33
[[http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber and Skerra,2005]] Schlehuber S, Skerra A.(2005)Lipocalins in drug discovery: from natural ligand-binding proteins to "anticalins".''Drug Discov Today''10(1):23-33
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[[http://www.pnas.org/content/103/15/6061 Yang et al.,2006]] Yang Y.,Sulpice R.,Himmelbach A.,Meinhard M.,Christmann A., Grill E. Fibrillin expression is regulated by abscisic acid response regulators and is involved in abscisic acid-mediated photoprotection ''PNAS'' 6061–6066
[[http://www.pnas.org/content/103/15/6061 Yang et al.,2006]] Yang Y.,Sulpice R.,Himmelbach A.,Meinhard M.,Christmann A., Grill E. Fibrillin expression is regulated by abscisic acid response regulators and is involved in abscisic acid-mediated photoprotection ''PNAS'' 6061–6066
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[[http://www.greenchicagoriver.com/ Homepage of the green chigargo river project]]
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[[http://www.ws.chemie.tu-muenchen.de/ Institute of Hydrochemistry and Chair for Analytical Chemistry ]]
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[[https://2008.igem.org/Team:Freiburg Homepage iGEM team Freiburg 2008]]
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[[http://www.ncbi.nlm.nih.gov/pubmed/11950570 Stoger et al.,2002]] Stoger E, Sack M, Fischer R, Christou P.(2002) Plantibodies: applications, advantages and bottlenecks ''Curr Opin Biotechnol.'' 13(2):161-6.
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[[http://www.ncbi.nlm.nih.gov/pubmed/20470236 Gan, 2010]] Gan TJ.(2010) Diclofenac: an update on its mechanism of action and safety profile ''Curr Med Res Opin''26(7):1715-31. doi: 10.1185/03007995.2010.486301
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[[http://www.pnas.org/content/96/5/1898.full.pdf Beste et al.,1999]]Beste G.,Schmidt F. S.,Stibora T.,Skerra A. (1999)''Proc. Natl. Acad. Sci. USA''Vol. 96, pp. 1898 –1903
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[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1724805/ Predel et al.,2004]] H Predel, R Koll, H Pabst, R Dieter, G Gallacchi, B Giannetti, M Bulitta, J Heidecker, and E Mueller(2004)Diclofenac patch for topical treatment of acute impact injuries: a randomised, double blind, placebo controlled, multicentre study ''Br J Sports Med'' 38(3): 318–323.
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[[http://onlinelibrary.wiley.com/doi/10.1002/prot.10497/abstract Korndörfer et. al. (2003)]]Korndörfer I.,Beste G.,Skerra A.(2003)Crystallographic analysis of an “anticalin” with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region ''Wiley''volume 53,issue 1,pages 121-129
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[[http://www.ncbi.nlm.nih.gov/pubmed/11276273 Moragan et al.,2001]] Morgan GJ Jr, Kaine J, DeLapp R, Palmer R (2001)Treatment of elderly patients with nabumetone or diclofenac: gastrointestinal safety profile ''J Clin Gastroenterol''32(4):310-4.
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[[http://www.ncbi.nlm.nih.gov/pubmed/21428697 Oakley,2006]] Oakley A.(2011)''Drug Metab Rev''43(2):138-51. doi: 10.3109/03602532.2011.558093
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[[http://www.ncbi.nlm.nih.gov/pubmed/15480981 Chan et al.,2004]] Chan FK, Hung LC, Suen BY, Wong VW, Hui AJ, Wu JC, Leung WK, Lee YT, To KF, Chung SC, Sung JJ.(2004) Celecoxib versus diclofenac plus omeprazole in high-risk arthritis patients: results of a randomized double-blind trial ''Gastroenterology'' 127(4):1038-43.
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[[http://www.ncbi.nlm.nih.gov/pubmed/16550164 McIlwain et al.,2006]] McIlwain CC, Townsend DM, Tew KD(2006) ''Oncogene''25(11):1639-48
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[[http://www.ncbi.nlm.nih.gov/pubmed/22168216 Pavelka,2012]] Pavelka K.(2012) A comparison of the therapeutic efficacy of diclofenac in osteoarthritis: a systematic review of randomised controlled trials ''Curr Med Res Opin.''28(1):163-78. doi: 10.1185/03007995.2011.649848.
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[[http://www.ncbi.nlm.nih.gov/pubmed/8770536 Hayes et al.,1995]]Hayes JD, Pulford DJ.(1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.''Crit Rev Biochem Mol Biol.''30(6):445-600
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[[http://www.ncbi.nlm.nih.gov/pubmed/16190224 Westerhoff et al. (2005)]] Westerhoff, P., Yoon, Y., Snyder, S., and Wert, E. (2005). Fate of endocrine- disruptor, pharmaceutical, and personal care product chemicals during simulated drinking water treatment processes. ''Environ Sci Technol'', 39(17):6649–63.
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[[http://www.ncbi.nlm.nih.gov/pubmed/20981235 Josephy, 2010]] Josephy PD.(2010)Genetic variations in human glutathione transferase enzymes: significance for pharmacology and toxicology.''Hum Genomics Proteomics'' 2010:876940. doi: 10.4061/2010/876940
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[[http://www.ncbi.nlm.nih.gov/pubmed/15984812 Huber et al., (2005)]] Huber, M. M., G ̈obel, A., Joss, A., Hermann, N., L ̈offler, D., McArdell, C. S., Ried, A., Siegrist, H., Ternes, T. A., and von Gunten, U. (2005). Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: a pilot study. ''Environ Sci Technol'', 39(11):4290–9.
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[[http://www.ncbi.nlm.nih.gov/pubmed/10971201 Hayes et al.,2000]]Hayes JD, Strange RC.(2000)Glutathione S-transferase polymorphisms and their biological consequences''Pharmacology'' 61(3):154-66
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[[http://www.ncbi.nlm.nih.gov/pubmed/12697241 Ternes et al., (2003)]] Ternes, T. A., Stu ̈ber, J., Herrmann, N., McDowell, D., Ried, A., Kampmann, M., and Teiser, B. (2003). Ozonation: a tool for removal of pharmaceuticals, contrast media and musk fragrances from wastewater? ''Water Res'', 37(8):1976–82.
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[[http://www.ncbi.nlm.nih.gov/pubmed/12563680 Fraser et al., 2003]]Fraser PA, Ding WZ, Mohseni M, Treadwell EL, Dooley MA, St Clair EW, Gilkeson GS, Cooper GS. (2003),''J Rheumatol''30(2):276-82
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[[http://www.ncbi.nlm.nih.gov/pubmed/12966990 Deng et al. (2003)]] Deng, A., Himmelsbach, M., Zhu, Q.-Z., Frey, S., Sengl, M., Buchberger, W., Niessner, R., and Knopp, D. (2003). Residue analysis of the pharmaceutical diclofenac in different water types using elisa and gc-ms. ''Environ Sci Technol'', 37(15):3422–9.
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[[https://2008.igem.org/Team:Freiburg Homepage iGEM team Freiburg 2008]]
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[[http://www.ncbi.nlm.nih.gov/pubmed/10051566 Beste et al., 1999]] Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold. ''PNAS'', 96(5):1898-903.
 +
 
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[[http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005]] Vopel S, Mühlbach H, Skerra A. (2005) Rational engineering of a fluorescein-binding anticalin for improved ligand affinity. ''Biol. Chem.'', 386(11):1097-104.
[[http://www.sciencedirect.com/science/article/pii/0965174895000909 La-Aied Prapanthadara et al.,1996]] La-Aied Prapanthadara,Surangchit Koottathe,Nongkran Promtet,Janet Hemingway†,Albert J., Ketterman (1996) Purification and characterization of a major glutathione S-transferase from the mosquito Anopheles dirus (Species B) ''Insect Biochemistry and Molecular Biology''Volume 26, Issue 3 Pages 277–285
[[http://www.sciencedirect.com/science/article/pii/0965174895000909 La-Aied Prapanthadara et al.,1996]] La-Aied Prapanthadara,Surangchit Koottathe,Nongkran Promtet,Janet Hemingway†,Albert J., Ketterman (1996) Purification and characterization of a major glutathione S-transferase from the mosquito Anopheles dirus (Species B) ''Insect Biochemistry and Molecular Biology''Volume 26, Issue 3 Pages 277–285
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[[http://www.ncbi.nlm.nih.gov/pubmed/11836138 Turusov et al.,2002 ]] Turusov V, Rakitsky V, Tomatis L.(2002)Dichlorodiphenyltrichloroethane (DDT): ubiquity, persistence, and risks''Environ Health Perspect.''110(2):125-8
[[https://2011.igem.org/Team:Caltech Homepage iGEM team Caltech 2011]]
[[https://2011.igem.org/Team:Caltech Homepage iGEM team Caltech 2011]]
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[[http://www.ncbi.nlm.nih.gov/pubmed/14715909 Ceulemans et al., 2004]] Ceulemans H, Bollen M.(2004) Functional diversity of protein phosphatase-1, a cellular economizer and reset button.''Physiol Rev.''84(1):1-39.
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[[http://www.ncbi.nlm.nih.gov/pubmed/21428697 Oakley,2006]] Oakley A.(2011)''Drug Metab Rev''43(2):138-51. doi: 10.3109/03602532.2011.558093
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[[http://www.ncbi.nlm.nih.gov/pubmed/17266553 Nekhai et al.,2007]] Nekhai S, Jerebtsova M, Jackson A, Southerland W.(2007)Regulation of HIV-1 transcription by protein phosphatase 1. Curr HIV Res. 5(1):3-9.
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[[http://www.ncbi.nlm.nih.gov/pubmed/16550164 McIlwain et al.,2006]] McIlwain CC, Townsend DM, Tew KD(2006)Glutathione S-transferase polymorphisms: cancer incidence and therapy ''Oncogene''25(11):1639-48
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[[http://www.ncbi.nlm.nih.gov/pubmed/8395566 Gong et al.,1993]] Gong CX, Singh TJ, Grundke-Iqbal I, Iqbal K.(1993),''J Neurochem''61(3):921-7.
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[[http://www.ncbi.nlm.nih.gov/pubmed/8770536 Hayes et al.,1995]]Hayes JD, Pulford DJ.(1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.''Crit Rev Biochem Mol Biol.''30(6):445-600
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[[http://www.nature.com/nature/journal/v376/n6543/abs/376745a0.html Goldberg et al.,2002]] Jonathan Goldberg, Hsien-bin Huang, Young-guen Kwon, Paul Greengard, Angus C. Nairn and John Kuriyan (2002)Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1 ''Nature 376''745 - 753 doi:10.1038/376745a0
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[[http://www.ncbi.nlm.nih.gov/pubmed/20981235 Josephy, 2010]] Josephy PD.(2010)Genetic variations in human glutathione transferase enzymes: significance for pharmacology and toxicology.''Hum Genomics Proteomics'' 2010:876940. doi: 10.4061/2010/876940
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[[http://www.ncbi.nlm.nih.gov/pubmed/24025782 Zhou et al.,2013]] Zhou Y, Chen Y, Yuan M, Xiang Z, Han X.(2013)''J Toxicol Sci.''38(5):661-70
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[[http://www.ncbi.nlm.nih.gov/pubmed/10971201 Hayes et al.,2000]]Hayes JD, Strange RC.(2000)Glutathione S-transferase polymorphisms and their biological consequences''Pharmacology'' 61(3):154-66
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[[https://2013.igem.org/Team:Dundee Homepage iGEM team Dundee 2013]]
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[[http://www.ncbi.nlm.nih.gov/pubmed/12563680 Fraser et al., 2003]]Fraser PA, Ding WZ, Mohseni M, Treadwell EL, Dooley MA, St Clair EW, Gilkeson GS, Cooper GS. (2003),Glutathione S-transferase M null homozygosity and risk of systemic lupus erythematosus associated with sun exposure: a possible gene-environment interaction for autoimmunity''J Rheumatol''30(2):276-82
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Latest revision as of 03:47, 29 October 2013


BioAccumulation

File:TUM13 General principle of accumulation of persistent chemicals along the food chain.jpg
Figure 1: General principle of accumulation of persistent chemicals along the food chain.

Bioaccumulation is a difficult concept to define. The [http://toxics.usgs.gov/definitions/bioaccumulation.html U.S. Environmental Protection Agency, 2010] defines bioaccumulation as a “general term describing a process by which chemicals are taken up by an organism either directly from exposure to a contaminated medium or by consumption of food containing the chemical”. The majority of definitions is aimed at the build up of persistent chemicals such as DDT, PBC or dioxins along the food chain (see figure 1), where the pollutants accumulate in predators until they become toxic. This can lead to the local and global extinction of species and health problems for humans. As most of these persistent chemicals are not biodegradable a different approach is necessary.

Figure 2: Principle of our bioaccumulation system.

Using our transgenic moos (Physcomitrella patens ) as a chassis, we can produce effectors for specific binding of pollutants. For the sustained removal of the target pollutant the effectors must be membrane-anchored, in contrast to the cytoplasmic or secretory localization of biodegrading effectors. The membrane association ensures the fixation of the pollutant to the membrane after successful binding. Internalisation of the effectors either results in metabolism or accumulation of the pollutant in the transgenic plant cell. In case of accumulation, controlled disposal of the plants in the filter would be necessary.

There is a broad range of natural as well as engineered binding proteins available. Natural binding proteins act as a model and initiator in design of new artificial binding proteins combining research in different fields of biotechnology. Lipocalins, natural binding proteins, as base and scaffold for the design of anticalins confirm the popularity of bioaccumulated proteins in red biotechnology http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber et al., 2005. The role of Fibrillin in the abscisic acid-mediated photoprotection shows an example of functioned bioaccumulation in a plant http://www.pnas.org/content/103/15/6061 Yang et al.,2007. The most commonly known binding proteins are antibodies which defend mammals against pathogens and toxins. Besides these natural binding proteins and anticalins there are more and more designed binding proteins such as Affibodies derived from the z-domain of the antibody-binding protein A (Ref) and DARPins that are based on an ankyrin scaffold. One research topic of the [http://www.ws.chemie.tu-muenchen.de chair of analytical chemistry of the TU Munich] is the depletion of algal toxin contaminated water by using selective biofilters based on plantibodies. Plantibodies are plant- produced and derived antibodies which can construct antibody-mediated pathogen resistance as well as change the plant phenotype by immunomodulation http://www.ncbi.nlm.nih.gov/pubmed/11950570 Stoger et al.,2002.

For our project we exemplarily choose three different binding proteins which each exploit different mechanisms to bind Proteins:

  • The anticalin fluA as an example for a binding protein.
  • The enzyme Gluthathione-S-Transferase as an example where the formation of covalent bonds between the target pollutant and another molecule (gluthatione) is catalized.
  • The enzyme Protein Phosphatase 1 as example for a target of a binding protein. Here we exploit the inhibitory binding of the pollutant to the enzyme.

To sketch the theoretical binding to a membrane anchored protein in our video, we created a short animation of the process using the molecularMaya software:

Bioaccumulation via binding proteins

Problem Example: Diclofenac

Are pharmaceuticals a blessing or a curse? Respective to the ecological consequences of diclofenac this task must be valued negative. Diclofenac which is a proven, nonsteroidal anti-imflammatory drug (NSAID) is used mostly in human medicine to treat a variety of acute and chronic pain and inflammatory conditions via inhibition of prostaglandin synthesis by inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)http://www.ncbi.nlm.nih.gov/pubmed/20470236 Gan,2010. Its necessity and importance in medicine research confirm e.g. the development of diclofenac patches for topical treatment of acute impact injuries http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1724805/ Predel et al.,2004 and the main studies about safety and efficiency of the analgesic, anti-inflammatory, and antipyretic properties of diclofenac http://www.ncbi.nlm.nih.gov/pubmed/11276273 Morgan et al.,2001http://www.ncbi.nlm.nih.gov/pubmed/15480981 Chan et al.,2004http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CHsQFjAI&url=http://www.ncbi.nlm.nih.gov/pubmed/22168216&ei=DLI5UqrPNJHSsgaL2YHAAQ&usg=AFQjCNFs7U5eWUrOKgcIuLly-afQqXpwTQ&sig2=ebhoYAq_NdG9v2nuxdB8BQ&bvm=bv.52288139,d.Yms Pavelka,2012. The possible resulting vicious circle (see Figure 1) shows the poisoning tragedy in 2005 which led to mass mortality and decline of vultures species especially Gyps bengalensis, Gyps indicus and Gyps tenuirostris across the Indian sub-continent. Based on the consumation of diclofenac by animals, vultures have an abnormally high intake in this drug by eating the cadavers of contaminated animals.

Current solutions

The fate of degradation products with their toxicity character and the development of antibiotic resistances plays a major role of the research on the effect of diclofenac in the aquatic environment. Because of the lack of analytical standards and the complex matrix of organic molecules in environmental samples, the degradation products have not yet been identified regarding molecular structure or concentration in aquatic fauna and flora.

The degree of the problem becomes visible in HPLC analysis, which shows in addition to the fast degradation of diclofenac over the irradiation time, a phytotoxicity increase of the increasing degradation product concentrations and a weak inhibition of cell growth.

To reduce the existence of PhACs such as diclofenac, one possibility is the usage of sewage treatment plants (STPs). Their removal efficiency depends on the sludge retention time and on the characteristics of the pharmaceutical molecule. Other strategies are the adding of an additional stage to the STP. Another approach is treated in the publications http://www.ncbi.nlm.nih.gov/pubmed/16190224 Westerhoff et al. (2005)http://www.ncbi.nlm.nih.gov/pubmed/15984812 Huber et al., (2005)http://www.ncbi.nlm.nih.gov/pubmed/12697241 Ternes et al., (2003) which confirm a high removal efficiency up to 95% for these additional stages. Currently developments are the nano-filtration, ozonation and power activated carbon. The importance of knowing the transformation products before applying a new technology is again demonstrated by the fact that ozonation reduces the parental substance diclofenac but cannot mineralize it.

To sum it up, biodegradation of diclofenac introduces several new problems as degradation products are toxic and pose new harms to the environment.

Bioaccumulation instead of Biodegradation

Figure 3: Structure of fluA(blue) with its ligand fluorescein(green) http://www.rcsb.org/pdb/explore/explore.do?structureId=1N0S source:PBD

As biodegradation does not conclusively solve the problem of diclofenac in the aquatic environment, we propose the utilisation of antibodies for accumulation of diclofenac, which would solve the problem of toxic degradation products.

Although an antibody against diclofenac has been developed http://www.ncbi.nlm.nih.gov/pubmed/12966990 Deng et al. (2003), in vivo verification of successful binding would not be straightforward. Therefore we aimed for a first proof of principle and decided to investigate another substance that is targetable by antibodies for which verification is feasible. One attractive option is the well-known fluorophore fluorescein which was used by the team Freiburg 2008. Fluorescein fluoresces until bound by the binding protein fluA, which makes characterization tractable. The covalent binding of fluA and flourescein is shown in Figure 3.

We submitted an improved version of the Freiburg Biobrick for fluA (see Table 1) and created a transgenic moss plants (PF-15) with the fluorescein binding anticalin fluA on the extracellular part of the receptor.

Producing engineered binding proteins for medical purpose in the red biotechnology is one of the main research fields in which anticalins play a major role. Lipocalins such as bilin-binding-protein BBP (from Pieris brassicae) can be used for generating molecular pockets with a diversity of shapes and for creating a stable receptor protein for a ligand of choice, so that development of binding proteins against nearly chemical structures with comparable size is possible. Hence it should be possible to target a great variety of pollutants by already available binding proteins.

Table 1: Variants of the fluorescein binding Anticalin FluA
Proteinvariant KD of FluA to fluorescein Literature reference BioBrick
FluA 152 nM http://www.ncbi.nlm.nih.gov/pubmed/10051566 Beste et al., 1999 <partinfo>BBa_K157004</partinfo>
FluA (R95K) 64 nM http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005 not available as BioBrick
FluA (R95K, A45I, S114T) 2 nM http://www.ncbi.nlm.nih.gov/pubmed/16307475 Vopel et al., 2005 <partinfo>BBa_K1159002</partinfo>

Glutathione-S-transferase

Problem: Dichlorodiphenyltrichloroethane (DDT)

Since the entry into force of the Stockholm Convention in 2004 the insecticide Dichlordiphenyltrichlorethan (DDT) is only allowed for the abatement of disease-carrying insects such as the malaria carrying Anopheles dirus mosquito http://www.sciencedirect.com/science/article/pii/0965174895000909 La-Aied Prapanthadara et al.,1996. This measure was the consequence to the devastating consequences for the ecosystem by the widespread use of DDT, which continuously grew due to it's strong insecticidal properties. Despite the regulation in 2004, DDT still poses an environmental threat today due to its stability and capacity to accumulate in tissue http://www.ncbi.nlm.nih.gov/pubmed/11836138 Turusov et al.,2002.

Current Solution

The simultaneous degradation of mixed insecticides like DDT can be achieved by mixed insecticide enriched isolated cultures in media and soil such as the mixed fungal population (white rot fungus Phanerochaete chrysosporium). The degradation efficiency of DDT can be enhanced by using carbon (mannitol) and nitrogen (sodium nitrate) in the liquid media. Based on the disadvantages which bring along the diverse strategies like chemical treatment, incineration, and landfills the development of the so called soil microflora method leads to a positive effect concerning to the detoxification of pesticides. The poor fungal growth in the media and concomitant biodegradation of insecticides is caused by toxicity exerted by high concentrations of the substrate which explains the low degradation at high concentrations.

Caltech 2011 Solution: Covalent binding to Gluthathion via Gluthathione-S-Transferase

Figure 4: Conjugation of GSH and a xenobiotic

In context to prove the bioaccumulation and to develop an effective methode of removing DDT, we envision the usage of the cytoplasmatic GST 1-1, also known as DDT Dehyrochlorinase. Along with the creation of transgenic Physcomitrella patens plants (PF-13) we used the biobrick from the Caltech iGEM team 2011. This team engineered bacteria which can degrade endocrine-disrupting chemicals such as DDT, synthetic estrogen in bodies of water to less toxic forms. Glutathion S-transferases (GSTs), an eukaryotic and prokaryotic phase II metabolic isozymes-family, catalyze the conjugation of reduced form of gluthatione (GSH, nucleophil) and xenobiotics (electrophil) to gluthatione-S-Conjugate via nucleophilic attack (see Figure 4).

Figure 5: Equilibrium reaction of gluthation

The consequence is an increased solubility of the conjugates which leads to the removal of xenobiotics in form of conjugates via vacuole enclosure. The action of the specific transporters and the steady supply of GSH in the equilibrium reaction (see Figure 5) are the limited factors of the detoxification reaction. Along with the detoxification and cell signaling function GST’s act as transport proteins, which gave GST the previous name ligandin. Table 2 shows the 3 different superfamilies with their characteristics.

File:TUM13 Structure3D of GST.gif
Figure 6: Molecular structure of Gluthation S-Transferase
Table 2: Superfamilies of GST with their characteristics
Superfamily of GTS Classes based upon their structure Sequence homology [%]
Cytosolic proteins alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega >40
Mitochondrial proteins kappa <25
microsomal (MAPEG= membrane-associated proteins in eicosanoid and glutathione metabolism) proteins subgroups I-IV <25
Figure 7: Reaction of monochlorobimane with GSH

Generally the three superfamilies differ mostly in structure and sequence as only the cytosolic and the mitochondrial superfamily have a thiorexin like domain in which the glutathione binding site (G-site) is located http://www.ncbi.nlm.nih.gov/pubmed/21428697 Oakley A.,2011. The helix alpha 2 is the most variable secondary structure. Y-GST is the subgroup which activates glutathione via using tyrosine residues. S/C-GST uses serine/cysteine residues. GST binds the substrate at the hydrophobic H-site of the enzyme and GSH at the hydrophilic G-site which together form the active site of the enzyme (see Figure 6). In research techniques GST is used as so called GST-tags for separation, elucidation of direct protein-protein interaction and purification of the GST-fusion protein mostly by pull-down assay. So targeting GST with molecule therapeutics represents GTS as an attractive target for drug discovery http://www.ncbi.nlm.nih.gov/pubmed/16550164 McIlwain et al.,2006.

A mammalian variant of GST, GSTP, plays a major role in cancer- development and potential drug/chemotherapeutic resistance in a majority of tumor cell lines: The inhibition of the pro-apoptotic pathway (JNK pathway) and the overexpression of GSTP in tumor cells lead to escape of apoptosis of the tumor cells mediated by non GSTP- substance-drugs http://www.ncbi.nlm.nih.gov/pubmed/8770536 Hayes et al.,1995 http://www.ncbi.nlm.nih.gov/pubmed/20981235 Josephy,2010 http://www.ncbi.nlm.nih.gov/pubmed/10971201 Hayes et al.,2000 http://www.ncbi.nlm.nih.gov/pubmed/12563680 Fraser et al.,2003. To avoid the time- and labor-intensive method HPLC following derivatization with 2-nitrobenzoic acid we used the common sensitive technique with monochlorobimane to measure GSH as a proof of principle. The adding of monochlorobimane to the culture medium leads to the conjugation of GSH to monochlorobimane catalyzed by DDT Dehyrochlorinase (see Figure 7). The GSH-monochlorobimane conjugate can be measured fluorometrically http://www.ncbi.nlm.nih.gov/pubmed/11038270 Kamencic et al.,2000.

Protein Phosphotase 1 – A molecular mop for Microcystin

The Problem: Algal Blooms

Figure 8: Eutrophication

Which phenomenon implicates the common name “Florida red tide”? This term is used in annually along Florida waters where species known as Karenia brevis leads to a red colored alga bloom along the Florida coast. Algal blooms are a worldwide environmental problem! This becomes evident with the appointment of 10.000 people for cleaning up the algal bloom in Beijing for the Olympic discipline “sailing regatta” in 2008. Depending on the cause, bacteria algal can bloom at concentrations of hundreds to thousands of cells per milliliter. An officially recognized threshold level isn’t public. Possible diverse factors which are the reason for the high affecting of bloom formation are listed followed:

  • Light intensity: cyanobacteria which form surface blooms have a higher tolerance concerning high light intensities. This leads to lower growth rate than other phytoplankton organisms.
  • Gas vacuoles: Gas vesicles give the cyanobacteria a lower density than water.
  • Growth rate: Slow growth rates require long water retention times to enable a bloom.
  • Phosphorous and nitrogen: A low ratio of these substance may favour a development of cyanobacteria blooms.
  • Temperature: maximum growth rates are attained by the most cyanobacteria above 25°C. Thus much bloom formation is during the summer.

A possible cascade which is caused by the release of nutrients in the aquatic ecosystems is shown in Figure 8. Another result confirms the spread and the persistence of cholera through algal blooms http://www.ncbi.nlm.nih.gov/pubmed/8155853 Epstein PR.,1993. Currently the so called harmful algal blooms are an additive for the detection of bacteria spread and their caused health effects to fauna and flora http://www.ncbi.nlm.nih.gov/pubmed/21135906 Ferrante et al.,2010 http://www.ncbi.nlm.nih.gov/pubmed/17298369 Barlaan EA et al.,2007. The reduction of the uptake of toxic methylmercury in freshwater food webs by algal blooms confirms a positive reverse of algal blooms http://www.ncbi.nlm.nih.gov/pubmed/11904388 Pickhardt PC. et al.,2002.

Current Solution to combat Algal blooms

It is well known that cyanobacteria produce most Toxins such as microcystin under species-specific conditions which are most favorable for their growth. As a result most studies concerning this topic try to tackle the problem from this point of view. For the investigated species, they found out that the toxin production increased under light optimum condition of the relative bacteria species and on low and high pH. The usage of zinc was necessary for the increase of growth and toxin production. Dependent on nitrogen fixing respectively non-nitrogen fixing species the nitrogen and phosphorous rate in media was unnecessary respectively necessary concerning to the toxin production. Evidences of the role of plasmid, multi-enzyme complexes and peptide synthetase gene involvement in toxin production were also confirmed: e.g. knockout experiments showed that peptide synthetase genes are responsible for microcysteine production.

Major problems are still the lack of knowledge regarding to occurrence and toxicity of cyanobacterial products like the diversity of LPS structures which cause allergic as well as toxic human health problems, the extremely weak scientific basis and poorly developed procedures for valuation on health effects. The TDI (tolerable daily intake) is one of the key features on which the toxicology studies are aimed. Based on analytic and sampling problems or inadequate scientific data safe practice guidelines can be assisted for the reducing of exposure.

The human exposure to the toxins is a major point to combat the human health risk. The WHO guideline values base upon a number of assumptions which change locally or nationally according to the present situation. To fill the lack of knowledge of parental variants the HPLC method is commonly used also for the determination of references. Current treatment processes used at water surface plants are coagulation, sand filtration, and clarification.

The negative site of these methods is the ineffective removal and destruction of cyanotoxins. To combat this problem treatment systems like carbon- and membrane filtration were developed. External and internal accumulation of toxins is another research topic since the inhibition of Mustard seedling development by of microcystein-LR in aquatic root solution.

Dundee 2013 Solution to combat Algal blooms

Due to the deleterious results from the toxin release of algal blooms many risk assesments were have been developed http://www.ncbi.nlm.nih.gov/pubmed/10499991 Schaeffer et al., 1999. The effects of microcystin for the aquatic and terrestrial ecosystem have been proven to include the modulation of proliferation, apoptosis and proliferation of spermatogenic cells in vivo http://www.ncbi.nlm.nih.gov/pubmed/24025782 Zhou et al.,2013, which makes it an attractive target for our project.

For the bioaccumulation of microcystin, it is possible to use the enzyme PP1. Mycrocystin can inhibit the function of PP1 by binding to it, this interaction of microcystin, in form of a heptapeptide structure, with three distinct regions of the PP1c is depicted in Figure 9. Hence PP1 can act as natural binding partner for mycrocystin.

As the Dundee iGEM team 2013 Team Dundee was already working on this subject we contacted them and they kindly provided us with the gene that encodes PP1. After converting the human PP1, to RFC 25 and constructing some expression plasmids we produced transgenic phytomitrella patens (PF-14) with PP1 and a biobrick of PP1 receptor.

References:

U.S. Geological Survey,2013 Definiton of Bioaccumulation

http://www.ncbi.nlm.nih.gov/pubmed/15676296 Schlehuber and Skerra,2005 Schlehuber S, Skerra A.(2005)Lipocalins in drug discovery: from natural ligand-binding proteins to "anticalins".Drug Discov Today10(1):23-33

http://www.pnas.org/content/103/15/6061 Yang et al.,2006 Yang Y.,Sulpice R.,Himmelbach A.,Meinhard M.,Christmann A., Grill E. Fibrillin expression is regulated by abscisic acid response regulators and is involved in abscisic acid-mediated photoprotection PNAS 6061–6066

http://www.ws.chemie.tu-muenchen.de/ Institute of Hydrochemistry and Chair for Analytical Chemistry

http://www.ncbi.nlm.nih.gov/pubmed/11950570 Stoger et al.,2002 Stoger E, Sack M, Fischer R, Christou P.(2002) Plantibodies: applications, advantages and bottlenecks Curr Opin Biotechnol. 13(2):161-6.

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