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Line 134:

case 12:

-

content.i = 12;

content.parents=[7];

-

content.childs=[13,14,15,16~~,37~~];

+

content.childs=[13; 14; 15; 16];

-

content.titleShort = "~~Ec.~~ ClpXP ~~system~~";

+

content.titleShort = "ClpXP, ssrA, SspB";

-

content.titleLong = "ClpXP Ec";

+

content.titleLong = "Short information about ClpXP, ssrA, SspB and how we used it in our projekt";

-

content.summary= "ClpXP Ex";

+

content.summary= "The ClpXP protease is able to degrade proteins with an ssrA tag. The SspB protein transfers this tagged protein to ClpXP.";

-

content.text= "ClpXP";

+

content.text= "We aimed to create a photosensitive protein degradation system which is useable in several types of cells with various proteins. In order to realize our system we decided to use the ClpXP protease, a protein break down system which is native to E. coli and mostly used to remove defective proteins and limit overall protein longevity <a href=#123>12.3</a> . ClpXP is an AAA+ Protease which consists of two subunits, the ClpP protein and the ClpX protein. The ClpX unit is assembled from a ring of subunits some of which are able to bind ATP. The ClpP protein consists of two rings encircling a central pore which functions as substrate binding site. When a protein is degraded the tagged substrate binds to the ClpX protein. It is consequently unfolded by the subunits through the hydrolysis of ATP. Finally the substrate is passed on to the ClpP unit which cleaves the amino bonds and releases amino acids. <a href=#121>12.1</a> The ClpXP protease can only break down proteins with a so called ssrA tag. This is a short amino acid sequence marking the protein for degradation by ClpXP. SsrA can be bound by the transporter protein SspB which increases the efficiency of degradation. The ssrA sequence has two functional parts. One part interacts with the ClpX protein of the ClpXP protease and one part is able to interact with SspB. A ssrA labled protein can either bind SspB or ClpXP in order to induce degradation. In normal E. coli strains this process not necessarily dependent on SspB but rather enhanced. A mutated form of ssrA (DAS+4) weakens the native binding between ClpX and ssrA and therefore increases the dependence of SspB. <a href=#122>12.2</a> The use of sspB induced protein degradation has been demonstrated with GFP <a href=#124>12.4</a> ,the neurotransmitter Arc <a href=#125>12.5</a> and Lac1, the Repressor of the lac operon.<a href=#126>12.6</a> <h3> SspB: </h3> SspB translocates the tagged protein to ClpXP and improves the affinity between ssrA and ClpXP and therefore mediates its breakdown.<a href=#127>12.7</a> To control degradation a split version of SspB can be used consisting of two domains: SspB[CORE] and SspB[XB] each of which cannot induce degradation on their own. To regain a functional construct for inducible degradation both were combined with a chemically inducible heterodimerisation system: FRB and FKBP12. In absence of rapamycin both parts are monomers and thus not functional. On addition of rapamycin FRB and FKBP12 form a dimer and consequently both SspB domains are in spatial proximity rendering them functional again.<a href=#129>12.9</a> Since there is a basic expression of SspB in regular E. coli strains a SspB deficient strain was used aiming to minimize constitutive degradation. In our project the SspB split-version was used but instead of rapamycin induced dimerization we used a LOV-ipaA - VinD1 mediated dimer in order to produce a functional split-SspB. <h2> References: </h2> <a name=121>12.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554 </a> <a name=122>12.2</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery[http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> <a name=123>12.3</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17291191[http://www.ncbi.nlm.nih.gov/pubmed/17291191]> The tmRNA System for Translational Surveillance and Ribosome Rescue </a> <a name=124>12.4</a> <a href=http://www.sciencedirect.com/science/article/pii/S1097276503002727[http://www.sciencedirect.com/science/article/pii/S1097276503002727]> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </a> <a name=125>12.5</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> <a name=126>12.6</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/]> Temperature dependence of ssrA-tag mediated protein degradation, Purcell et al, 2012, Journal of Biological Engineering, PMID: 22824000 </a> <a name=127>12.7</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/14967151[http://www.ncbi.nlm.nih.gov/pubmed/14967151]> Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </a> <a name=128>12.8</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> <a name=129>12.9</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/]> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> ";

content.type="Background";

break;

@@ Line 134: / Line 134: @@
 case 12:
 content.i = 12;
 content.parents=[7];
-content.childs=[13,14,15,16,37];
+content.childs=[13; 14; 15; 16];
-content.titleShort = "Ec. ClpXP system";
+content.titleShort = "ClpXP, ssrA, SspB";
-content.titleLong = "ClpXP Ec";
+content.titleLong = "Short information about ClpXP, ssrA, SspB and how we used it in our projekt";
-content.summary= "ClpXP Ex";
+content.summary= "The ClpXP protease is able to degrade proteins with an ssrA tag. The SspB protein transfers this tagged protein to ClpXP.";
-content.text= "ClpXP";
+content.text= "We aimed to create a photosensitive protein degradation system which is useable in several types of cells with various proteins. In order to realize our system we decided to use the ClpXP protease, a protein break down system which is native to E. coli and mostly used to remove defective proteins and limit overall protein longevity <sup><a href=#123>12.3</a></sup> </br> . ClpXP is an AAA+ Protease which consists of two subunits, the ClpP protein and the ClpX protein. The ClpX unit is assembled from a ring of subunits some of which are able to bind ATP. The ClpP protein consists of two rings encircling a central pore which functions as substrate binding site. When a protein is degraded the tagged substrate binds to the ClpX protein. It is consequently unfolded by the subunits through the hydrolysis of ATP. Finally the substrate is passed on to the ClpP unit which cleaves the amino bonds and releases amino acids. <sup><a href=#121>12.1</a></sup> </br> </br> The ClpXP protease can only break down proteins with a so called ssrA tag. This is a short amino acid sequence marking the protein for degradation by ClpXP. SsrA can be bound by the transporter protein SspB which increases the efficiency of degradation. </br> The ssrA sequence has two functional parts. One part interacts with the ClpX protein of the ClpXP protease and one part is able to interact with SspB. </br> A ssrA labled protein can either bind SspB or ClpXP in order to induce degradation. In normal E. coli strains this process not necessarily dependent on SspB but rather enhanced. A mutated form of ssrA (DAS+4) weakens the native binding between ClpX and ssrA and therefore increases the dependence of SspB. <sup><a href=#122>12.2</a></sup> </br> </br> The use of sspB induced protein degradation has been demonstrated with GFP <sup><a href=#124>12.4</a></sup> ,the neurotransmitter Arc <sup><a href=#125>12.5</a></sup> and Lac1, the Repressor of the lac operon.<sup><a href=#126>12.6</a></sup> </br> </br> <h3> <b>SspB:</b> </h3> SspB translocates the tagged protein to ClpXP and improves the affinity between ssrA and ClpXP and therefore mediates its breakdown.<sup><a href=#127>12.7</a></sup> </br> To control degradation a split version of SspB can be used consisting of two domains: SspB[CORE] and SspB[XB] each of which cannot induce degradation on their own. To regain a functional construct for inducible degradation both were combined with a chemically inducible heterodimerisation system: FRB and FKBP12. In absence of rapamycin both parts are monomers and thus not functional. On addition of rapamycin FRB and FKBP12 form a dimer and consequently both SspB domains are in spatial proximity rendering them functional again.<sup><a href=#129>12.9</a></sup> </br> Since there is a basic expression of SspB in regular E. coli strains a SspB deficient strain was used aiming to minimize constitutive degradation. </br> In our project the SspB split-version was used but instead of rapamycin induced dimerization we used a LOV-ipaA - VinD1 mediated dimer in order to produce a functional split-SspB. </br> <h2> <b>References:</b> </h2> </br> <a name=121>12.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554 </a> </br> <a name=122>12.2</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery[http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> <a name=123>12.3</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17291191[http://www.ncbi.nlm.nih.gov/pubmed/17291191]> The tmRNA System for Translational Surveillance and Ribosome Rescue </a> </br> <a name=124>12.4</a> <a href=http://www.sciencedirect.com/science/article/pii/S1097276503002727[http://www.sciencedirect.com/science/article/pii/S1097276503002727]> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </a> </br> <a name=125>12.5</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> <a name=126>12.6</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/]> Temperature dependence of ssrA-tag mediated protein degradation, Purcell et al, 2012, Journal of Biological Engineering, PMID: 22824000 </a> </br> <a name=127>12.7</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/14967151[http://www.ncbi.nlm.nih.gov/pubmed/14967151]> Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </a> </br> <a name=128>12.8</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> </br> <a name=129>12.9</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/]> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> </br>";
 content.type="Background";
 break;

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