Difference between revisions of "Team:SCUT-China/Description"

 
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     <h1 id="projectTitle">Description</h1>
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             <h2>Content</h2>
 
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             <h3>1. Overview</h3>
 
             <h3>1. Overview</h3>
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             <h4>3.2 Silence the PDE5A</h4>
 
             <h4>3.2 Silence the PDE5A</h4>
 
             <h4>3.3 On-Off: Hypoxia-Inducible Promoter</h4>
 
             <h4>3.3 On-Off: Hypoxia-Inducible Promoter</h4>
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        </div>-->
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        <div class="part">
 +
            <h3 style="color:#00b4ed">1. Overview</h3>
 +
            <p>According to data from the World Health Organization (WHO), cardiovascular diseases are the leading causes of death globally. Cyclic guanosine monophosphate (cGMP) is a critical second messenger molecule. One of its many complex functions is to transduce nitric-oxide (NO) and natriuretic-peptide-coupled signaling and remit myocardial dysfunctions by relaxing blood vessels. This summer, we tried to use synthetic biology to modify the cGMP metabolic pathway in a human cell line. We hope that our work could provide basis for a future gene therapy using synthetic biology.</p>
 +
            <img src="https://static.igem.org/mediawiki/2015/4/49/2015-SCUT-China-home-overview.png" class="img" />
 +
          <div id="Note" class="part"> 
 +
            <p>NO：nitric oxide,      sGC：soluble guanylate cyclase,    PKG：protein kinase G  </p>
 +
            <p>PDE5A：Phosphodiesterase-5A,  HIF： hypoxia inducible factor-1,  HRE：  hypoxia responsive element</p>
 +
          </div>
 +
            <p>Increased understanding of the molecular events operating in the NO-sGC-cGMP axis has stimulated therapeutic targeting of the pathway in human diseases. Soluble guanylate cyclase (sGC) is an enzyme that synthesizes cGMP from GTP and is found as a heterodimer of α and β subunits. BBa_K1720000 and BBa_K1720001 contain the full coding sequences of the α and β subunits of human sGC, respectively. Co-expression of the two parts increases sGC activity and cGMP synthesis in the cell. However, elevated cellular level of cGMP may lead to feed-back expression of PDE5A, a phosphodiesterase that degrades cGMP. Creation of novel circuit that consists of U6 promoter followed by PDE5A shRNA may enhance the function of our parts. To achieve controllable up-regulation of cGMP level in the cell, we also designed a hypoxia-inducible operon, HRE, as a switch to up-regulate cGMP only in hypoxia situation.</p>
 
         </div>
 
         </div>
 
         <div class="part">
 
         <div class="part">
−
             <h3>1. Overview</h3>
+
             <h3 style="color:#06afe8">2. Background</h3>
−
            <p>According to the data from WHO, cardiovascular diseases are the main leading cause of death globally. Cyclic guanosine monophosphate (cGMP) is a critical second messenger molecule.It can transduce nitric-oxide and natriuretic-peptide-coupled signaling and remit the myocardial hypertrophy by relaxing the blood vessels. This summer, we tried to use synthetic biology to modify the cGMP metabolic pathway in a human cell line. We hope that our work would provide the proof of principle for future gene therapy.</p>
+
           
−
             <img src="image/2015-SCUT-China-homeoverview.png" class="img" />
+
            <div class="part">
−
             <p>Soluble guanylate cyclase (sGC) is an enzyme that synthesize cGMP from GTP. We up-regulate sGC by overexpressing its α and β subunits  in a mammalian cell line. However, elevated levels of cGMP leads to the feed-back expression of PDE5a, a cGMP-specific phosphodiesteras that degrades cGMP. Thus, we further modified the pathway by silencing the PDE5a. To achieve controllable up-regulation of cGMP level in the cell, we designed a hypoxia-inducible operon, HRE, as a switch to up regulate cGMP only in hypoxia situation.</p>
+
<img class="img" src="https://static.igem.org/mediawiki/2015/d/dc/2015-SCUT-China-project_description_background1.jpeg"  />
 +
<div id="Note" class="part"> 
 +
<p>   The 10 leading causes of death in the world by percentage from WHO</div>
 +
<a class="source_link" href=" http://www.who.int/mediacentre/factsheets/fs310/en/"> (Sources: http://www.who.int/mediacentre/factsheets/fs310/en/)</a></p></div>
 +
            <p>Ischemic and hypertensive heart disease were the leading causes of death in the world over the past decade. Over 7.4 million people were dead of these diseases in 2012 and the number is increasing. Patients and their families suffer from the diseases. It also left a heavy burden to government and society. This summer, we tried to solve the problem with our knowledge in synthetic biology.</p>
 +
             <p>The discovery of Nitric oxide (NO) as an endogenous signaling molecule and as a mediator of the cardiovascular effects was acknowledged by the Nobel Prize in Physiology/Medicine in 1998. NO activates sGC by binding to its prosthetic heme group and thereby catalyzing the synthesis of cGMP. As a critical second messenger molecule, cGMP regulates many vital homeostatic mechanisms, i.e. endothelial cell permeability, vascular smooth muscle contractility and cardiomyocyte hypertrophy. </p>
 +
 
 +
             <p>Understanding of the NO-sGC-cGMP pathway has initiated significant translational interest, but this was almost exclusively embodied by the use of NO donors and PDE5 inhibitors. NO donors are not ideal for chronic treatment of heart diseases because of partial patient response, development of tolerance over time and short retention in the plasma. The use of PDE inhibitors is also likely to be limited as the efficacy of such molecules is dependent on endogenous cGMP generation. We therefore thought that it may be beneficial to ‘think outside the box’ to target the NO-sGC-cGMP axis using molecular medicines.</p>
 
         </div>
 
         </div>
−
          
+
         <div class="part">
−
        <div class="part">
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             <h3 style="color:#08ade5">3. Project</h3>
−
             <h3>2. Background</h3>
+
            <h4 style="color:#0ea9e2">3.1 Over Expression of sGC</h4>
−
        <img src="image/2015-SCUT-China-homeoverview1.png" class="img" />  
+
            <p>Dysfunction of NO signaling is related to many pathological disorders, such as myocardial hypertrophy, arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. Soluble guanylate cyclase (sGC) is a critical enzyme in NO-sGC-cGMP pathway, which catalyzes the production of cGMP from GTP. In this project, we aimed to up-regulate the concentration of cGMP by the overexpression of sGC to enhance the function of NO signaling pathway. SGC is a heterodimeric protein, contains two subunits: α and β. Both α and β subunits have a few different isoforms. We overexpressed the α3 (GUCY1A3) and β3 (GUCY1B3) isoforms because they are the most abundant forms in the cardiovascular system. </p>
−
    <img src="image/2015-SCUT-China-homeoverview2.png" class="img" />
+
            <div class="imgContent"> <img style="width:30%;" src="https://static.igem.org/mediawiki/2015/3/39/2015-SCUT-China-project-description1.png" class="img" />
−
    <img src="image/2015-SCUT-China-homeoverview3.png" class="img" />
+
            <div id="Note" class="part">               
−
<a href=" http://www.who.int/mediacentre/factsheets/fs310/en/"> (Sources: http://www.who.int/mediacentre/factsheets/fs310/en/)</a>
+
              <p> Crystal structure of the heterodimeric catalytic domain of wild-type human soluble guanylate cyclase.</p>
−
            <p>From the data of WHO in 2012, it’s obvious that ischemic heart disease was the main leading cause of death in the world over the past decade. During this period, the figure of ischemic heart disease kept on rising and it rose to 13.2% in 2012. A large number of patients suffered from ischemic heart disease every year. The increasing number of patients also left a heavy burden to government and society. In order to change this serious situation, something must be done to stop the rapid growth tendency of ischemic heart disease. Therefore, this summer we tried to do something to protect against this disease. </p>
+
            </div>
 +
                <a class="source_link" href=" http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1#"> (Sources:http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1# )</a>  
 
         </div>
 
         </div>
−
  <div class="part">
+
       
−
             <h3>3. Project</h3>
+
 
−
<h4>3.1 Over Expression of sGC</h4>
+
        <div class="part">
−
<p>Soluble guanylate cyclase (sGC) a critical enzyme in nitric oxide (NO)—soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway. This pathway serves an important physiologic role in vascular tissues,including remission of myocardial hypertrophy. SGC catalyzes GMP to form cGMP ,a second messenger molecule that transduces nitric-oxide and natriuretic-peptide-coupled signaling. Dysfunction of NO signaling results in many pathological disorders, such as myocardial hypertrophy , arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. In our project we aimed to up regulate the concentration of cGMP by the overexpression of sGC to reestablish the function of NO signaling pathway. SGC is a heterodimeric protein, containing 2 subunits: alpha and beta. However, both alpha and beta subunit has a few different isoforms. In our project we overexpressed the alpha3 and beta3 isoforms because they are abundant in cardiovascular system. </p>
+
             <h4 style="color:#11a7e2">3.2 Silence the PDE5A</h4>
−
      <div class="imgContent"> <img src="image/2015-SCUT-China-sGC.png" class="img" /><p> Crystal structure of the heterodimeric catalytic domain of wild-type human soluble guanylate cyclase.</p><a href=" http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1#"> (Sources:http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1#)</a> </div>
+
            <p>cGMP is catabolized to 5’GMP by specific members of the phosphodiesterase (PDE) superfamily. Our goal to increase cGMP synthesis by overexpressing sGC was achieved, but this may also lead to increased cGMP catabolism through the up-regulation of PDE5A. Thus, we created novel circuit consisting U6 promoter followed by PDE5A shRNA to enhance the effects of sGC overexpression. </p>
 +
            <div class="imgContent"> <img src="https://static.igem.org/mediawiki/2015/0/02/2015-SCUT-China-project_silence111.png" class="img" /> </div>
 
         </div>
 
         </div>
 +
        <div class="part">
 +
            <h4 style="color:#1c9eda">3.3 On-Off: Hypoxia-Inducible Promoter</h4>
 +
            <p>Absolutely,the level of cGMP will be up regulated when we enhance cGMP synthesis and block its degradation at the same time.If the devices “overexpress sGC” and “silence PDE5A” work in myocardium cells overtime,it may lead several bad effects we don’t expect.To achieve controllable up-regulation of cGMP level in the cell, we also need a switch to up regulate cGMP only in ischemia or hypoxia situation. </p>
 +
            <div class="imgContent"> <img src="https://static.igem.org/mediawiki/2015/e/e2/2015-SCUT-China-project_description_promoter11.png" class="img" /> </div>
 +
            <p>We found that in mammalian cells, there is already a hypoxia-inducible system. The hypoxia-inducible factor (HIF) activates transcription via binding to highly variable hypoxia-responsive elements (HREs) that are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. Space between HBSs, distance from the core promoter, and orientation of HBSs are all connected with hypoxia-responsive activity. Following these rules, we design a HRE sequence and inserted it into the CMV promoter, ahead of the TATA box. We name the reconstructed CMV promoter as hypoxia-inducible promoter. To examine its function, we test an original CMV promoter submitted by the team Freiburg in 2012 as negative control,<a style="color:#343434" href="http://parts.igem.org/Part:BBa_K747096">BBa_k747096</a>.Thus, we improved the characterization of a previously existing basic part.</p>
 +
            <div class="imgContent"> <imgsrc="https://static.igem.org/mediawiki/2015/f/ff/2015-SCUT-China-project_description_promoter22.png" class="img" /> </div>
 +
        </div>
 +
    </div>
  
 +
<div id="reference" class="part">
 +
<h3 style="color:#1c9eda">4. Referrence</h3>
 +
            <p>1. Gheorghiade M, Marti CN, Sabbah HN, Roessig L, Greene SJ, Bohm M, Burnett JC, Campia U, Cleland JG, Collins SP et al: Soluble guanylate cyclase: a potential therapeutic target for heart failure. Heart failure reviews 2013, 18(2):123-134.</p>
 +
<p>2. Sharina IG, Cote GJ, Martin E, Doursout MF, Murad F: RNA splicing in regulation of nitric oxide receptor soluble guanylyl cyclase. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 2011, 25(3):265-274.</p>
 +
<p>3. Marinko M, Novakovic A, Nenezic D, Stojanovic I, Milojevic P, Jovic M, Ugresic N, Kanjuh V, Yang Q, He GW: Nicorandil directly and cyclic GMP-dependently opens K channels in human bypass grafts. Journal of pharmacological sciences 2015.</p>
 +
<p>4. Ikoma E, Tsunematsu T, Nakazawa I, Shiwa T, Hibi K, Ebina T, Mochida Y, Toya Y, Hori H, Uchino K et al: Polymorphism of the type 6 adenylyl cyclase gene and cardiac hypertrophy. J Cardiovasc Pharmacol 2003, 42:S27-S32.</p>
 +
<p>5. Hodges GJ, Gros R, Hegele RA, Van Uum S, Shoemaker JK, Feldman RD: Increased blood pressure and hyperdynamic cardiovascular responses in carriers of a common hyperfunctional variant of adenylyl cyclase 6. The Journal of pharmacology and experimental therapeutics 2010, 335(2):451-457.</p>
 +
<p>6. Vandamme J, Castermans D, Thevelein JM: Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation. Cellular signalling 2012, 24(8):1610-1618.</p>
 +
<p>7. Gros R, Van Uum S, Hutchinson-Jaffe A, Ding Q, Pickering JG, Hegele RA, Feldman RD: Increased enzyme activity and beta-adrenergic mediated vasodilation in subjects expressing a single-nucleotide variant of human adenylyl cyclase 6. Arteriosclerosis, thrombosis, and vascular biology 2007, 27(12):2657-2663.</p>
 +
<p>8. Roth DM: Adenylyl Cyclase Increases Survival in Cardiomyopathy. Circulation 2002, 105(16):1989-1994.</p>
 +
<p>9. Kuhn M: Structure, regulation, and function of mammalian membrane guanylyl cyclase receptors, with a focus on guanylyl cyclase-A. Circulation Research 2003, 93(8):700-709.</p>
 +
<p>10. Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, Bedja D, Gabrielson KL, Wang Y, Kass DA: Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nature 2005.</p>
 +
<p>11. Kukreja RC, Salloum FN, Das A: Cyclic Guanosine Monophosphate Signaling and Phosphodiesterase-5 Inhibitors in Cardioprotection. Journal Of the American College Of Cardiology 2012, 59(22):1921-1927.</p>
 +
<p>12. Kuhn M: CARDIOLOGY A big-hearted molecule. Nature 2015, 519(7544):416-417.</p>
 +
<p>13. Singh N, Patra S: Phosphodiesterase 9: insights from protein structure and role in therapeutics. Life sciences 2014, 106(1-2):1-11.</p>
 +
<p>14. Lee YC, Martin E, Murad F: Human recombinant soluble guanylyl cyclase: expression, purification, and regulation. Proceedings of the National Academy of Sciences of the United States of America 2000, 97(20):10763-10768.</p>
 +
<p>15. Li L, Haider HK, Wang L, Lu G, Ashraf M: Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction. American Journal Of Physiology-Heart And Circulatory Physiology 2012, 302(10):H2112-H2121.</p>
 +
<p>16. Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP et al: Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease. Nature 2015, 519(7544):472-476.</p>
 +
<p>17. Wobst J, Rumpf PM, Dang TA, Segura-Puimedon M, Erdmann J, Schunkert H: Molecular Variants of Soluble Guanylyl Cyclase Affecting Cardiovascular Risk. Circulation Journal 2015, 79(3):463-469.</p>
 +
<p>18. Unwalla HJ, Li HT, Bahner I, Li MJ, Kohn D, Rossi JJ: Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. Journal of virology 2006, 80(4):1863-1873.
 +
</p>
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Latest revision as of 00:52, 19 September 2015

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Description

1. Overview

According to data from the World Health Organization (WHO), cardiovascular diseases are the leading causes of death globally. Cyclic guanosine monophosphate (cGMP) is a critical second messenger molecule. One of its many complex functions is to transduce nitric-oxide (NO) and natriuretic-peptide-coupled signaling and remit myocardial dysfunctions by relaxing blood vessels. This summer, we tried to use synthetic biology to modify the cGMP metabolic pathway in a human cell line. We hope that our work could provide basis for a future gene therapy using synthetic biology.

NO：nitric oxide, sGC：soluble guanylate cyclase, PKG：protein kinase G

PDE5A：Phosphodiesterase-5A, HIF： hypoxia inducible factor-1, HRE： hypoxia responsive element

Increased understanding of the molecular events operating in the NO-sGC-cGMP axis has stimulated therapeutic targeting of the pathway in human diseases. Soluble guanylate cyclase (sGC) is an enzyme that synthesizes cGMP from GTP and is found as a heterodimer of α and β subunits. BBa_K1720000 and BBa_K1720001 contain the full coding sequences of the α and β subunits of human sGC, respectively. Co-expression of the two parts increases sGC activity and cGMP synthesis in the cell. However, elevated cellular level of cGMP may lead to feed-back expression of PDE5A, a phosphodiesterase that degrades cGMP. Creation of novel circuit that consists of U6 promoter followed by PDE5A shRNA may enhance the function of our parts. To achieve controllable up-regulation of cGMP level in the cell, we also designed a hypoxia-inducible operon, HRE, as a switch to up-regulate cGMP only in hypoxia situation.

2. Background

The 10 leading causes of death in the world by percentage from WHO

(Sources: http://www.who.int/mediacentre/factsheets/fs310/en/)

Ischemic and hypertensive heart disease were the leading causes of death in the world over the past decade. Over 7.4 million people were dead of these diseases in 2012 and the number is increasing. Patients and their families suffer from the diseases. It also left a heavy burden to government and society. This summer, we tried to solve the problem with our knowledge in synthetic biology.

The discovery of Nitric oxide (NO) as an endogenous signaling molecule and as a mediator of the cardiovascular effects was acknowledged by the Nobel Prize in Physiology/Medicine in 1998. NO activates sGC by binding to its prosthetic heme group and thereby catalyzing the synthesis of cGMP. As a critical second messenger molecule, cGMP regulates many vital homeostatic mechanisms, i.e. endothelial cell permeability, vascular smooth muscle contractility and cardiomyocyte hypertrophy.

Understanding of the NO-sGC-cGMP pathway has initiated significant translational interest, but this was almost exclusively embodied by the use of NO donors and PDE5 inhibitors. NO donors are not ideal for chronic treatment of heart diseases because of partial patient response, development of tolerance over time and short retention in the plasma. The use of PDE inhibitors is also likely to be limited as the efficacy of such molecules is dependent on endogenous cGMP generation. We therefore thought that it may be beneficial to ‘think outside the box’ to target the NO-sGC-cGMP axis using molecular medicines.

3. Project

3.1 Over Expression of sGC

Dysfunction of NO signaling is related to many pathological disorders, such as myocardial hypertrophy, arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. Soluble guanylate cyclase (sGC) is a critical enzyme in NO-sGC-cGMP pathway, which catalyzes the production of cGMP from GTP. In this project, we aimed to up-regulate the concentration of cGMP by the overexpression of sGC to enhance the function of NO signaling pathway. SGC is a heterodimeric protein, contains two subunits: α and β. Both α and β subunits have a few different isoforms. We overexpressed the α3 (GUCY1A3) and β3 (GUCY1B3) isoforms because they are the most abundant forms in the cardiovascular system.

Crystal structure of the heterodimeric catalytic domain of wild-type human soluble guanylate cyclase.

(Sources:http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1# )

3.2 Silence the PDE5A

cGMP is catabolized to 5’GMP by specific members of the phosphodiesterase (PDE) superfamily. Our goal to increase cGMP synthesis by overexpressing sGC was achieved, but this may also lead to increased cGMP catabolism through the up-regulation of PDE5A. Thus, we created novel circuit consisting U6 promoter followed by PDE5A shRNA to enhance the effects of sGC overexpression.

3.3 On-Off: Hypoxia-Inducible Promoter

Absolutely,the level of cGMP will be up regulated when we enhance cGMP synthesis and block its degradation at the same time.If the devices “overexpress sGC” and “silence PDE5A” work in myocardium cells overtime,it may lead several bad effects we don’t expect.To achieve controllable up-regulation of cGMP level in the cell, we also need a switch to up regulate cGMP only in ischemia or hypoxia situation.

We found that in mammalian cells, there is already a hypoxia-inducible system. The hypoxia-inducible factor (HIF) activates transcription via binding to highly variable hypoxia-responsive elements (HREs) that are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. Space between HBSs, distance from the core promoter, and orientation of HBSs are all connected with hypoxia-responsive activity. Following these rules, we design a HRE sequence and inserted it into the CMV promoter, ahead of the TATA box. We name the reconstructed CMV promoter as hypoxia-inducible promoter. To examine its function, we test an original CMV promoter submitted by the team Freiburg in 2012 as negative control,BBa_k747096.Thus, we improved the characterization of a previously existing basic part.

4. Referrence

1. Gheorghiade M, Marti CN, Sabbah HN, Roessig L, Greene SJ, Bohm M, Burnett JC, Campia U, Cleland JG, Collins SP et al: Soluble guanylate cyclase: a potential therapeutic target for heart failure. Heart failure reviews 2013, 18(2):123-134.

2. Sharina IG, Cote GJ, Martin E, Doursout MF, Murad F: RNA splicing in regulation of nitric oxide receptor soluble guanylyl cyclase. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 2011, 25(3):265-274.

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