To achieve this we decided to look through the registry at existing promoter which would select lycopene expression only in 'high stress' conditions which mimic the imbalance of stress within the cell. Hence, we decided to combine lycopene with a stress sensitive promoter which would only release lycopene when cells are stressed. This promoter was NarK. We are very grateful to Aubrey for his insight and enthusiasm for our project, and also thankful that he took time to speak with our team and influence our project in a positive way.


To watch our conversation click here.

Early in our discussion of what we could target in ageing, we spoke to various academics, including Dr. Max Yun, a Senior Research Associate in the UCL’s Division of Biosciences who recommended a paper to us - ‘The Hallmarks of Ageing’. This highlighted not only is ageing a complex, multi-faceted problem, but also that it wouldn’t be possible to target all hallmarks, instead, we chose to focus on reactive oxygen species (ROS) which underpin some of the hallmarks (1) of ageing.

For instance, endogenous damage caused by ROS leads to DNA lesions including telomerase shortening(1). These lesions essentially lead to genomic instability (1). Ageing in this instance is caused by imbalance between DNA damage and DNA repair, for instance insufficient repair mechanism or excessive DNA damage promotes ageing (1).

Oxidative stress can also cause proteins to unfold and aggregate, again leading to ageing (1). We found greater strength in this argument when we were shown data from research undertaken at Imperial University showing oxidative stress to have a detrimental impact upon protein stability. The consequence of this is in turn reflected in protein activity and hence ageing. A description of these results is included below.

We have been researching the link between protein aggregation and oxidative stress. By using Dynamic Light Scattering (DLS), Yash was able to see that the size of a protein (monoclonal antibody) changed in the presence of oxidative stress. Under oxidative stress the protein becomes unstable and either undergoes a conformational change forming an unstable intermediate with a radius of a bit more than 1nm (as seen in image 2), or it aggregates (seen as the 2nd peak (at ~100-1000nm of the 2nd picture). 5mM of Hydrogen Peroxide was used to induce oxidative stress.

Particle Size Distribution with Oxidative Stress

 

We also did some experimentation using Self-Interaction Chromatography (SIC). As shown below, without oxidative stress we get a nice peak and the area under the peak can be used to calculate the 2nd Osmotic Virial Coefficient (B2). B2 is a dimensionless no. that indicates protein stability. A positive B2 means that the proteins particles repel each other and will tend not to aggregate, but a negative B2 means that the protein is unstable, as its particles have a net attractive interaction and will aggregate. Without oxidative stress, the protein is stable with a B2 of 0.66.

However, from the graph below, which shows the protein when it's exposed to oxidative stress, oxidative stress causes particle-particle interaction to become very high and very attractive. This is why the peak goes on for a while because particles are taking more and more time interacting with each other and the column attracts the mAb and heavily disrupts the flow of it. B2 with oxidative stress is -21, meaning that the protein is very unstable.

In conclusion, oxidative stress has a very significant impact on protein stability. As you age and there is more oxidative stress in the body, the proteins in our cells which are essential to healthy functioning become unstable/aggregate, thereby leading to a loss of activity. This loss of activity of the essential proteins leads to the deterioration of health, so targeting oxidative stress to promote healthy ageing can be an effective strategy.

Clearly the impact of oxidative stress upon protein structure is significant and since structure and function are inexplicably linked the functionality of the proteins are likely to be hindered too. Thus, we are looking towards preventative methods that will lessen the amount of oxidative stress that proteins are exposed to.