Team:Queens/Results

Results

The following summarizes the results of the various aspects of this year's QGEM Project.

Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.

Fig. 1 SAA expression and secretion by E. coli cells. Cultures of E. coli cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein.

Our E. coli cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future.

References:

Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. New York Academy of Sciences. 82:183-189.
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. Journal of Biological Chemistry. Vol. 274, 11:7172-7181.

Ideally, our effector system should only begin to produce the effectors once a threshold density of E.coli cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque.

In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects.

1. Serum Amyloid A

SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression.

However, the clinical effectiveness of SAA is still in doubt for several reasons:

    * Not all atherosclerotic plaques are associated with cholesterol.
    * The body can naturally produce SAA at an astoundingly high rate, given the correct signals.
    * We are unsure if the molecule will reach the area of the plaque it is effective in.
    * We are unsure if the body produces enough HDL to clear atherosclerotic plaques.

2. Heme Oxygenase 1 (HO-1)

The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture.

3. Atrial Natriuretic Peptide (ANP)

ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.

After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.

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