Team:SCUT-China/Description

Loading

• 
• HOME
• TEAM
  • Team Members
  • Attribution
• PROJECT
  • Description
  • Experiments & Results
  • Protocols
  • Notebook
• PARTS
• COLLABORATIONS
• HUMAN PRACTICES
  • Overview
  • Hospital Interview
  • Seminar & Conference
  • Data Analysis
  • Law & Regulation
• SAFETY
• MODELING

Project description

Content

1. Overview

2. Background

3. Project

3.1 Over Expression of sGC

3.2 Silence the PDE5A

3.3 On-Off: Hypoxia-Inducible Promoter

1. Overview

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.

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.

2. Background

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

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.

3. Project

3.1 Over Expression of sGC

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.

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#)