Preliminary data

With Halloween on the horizon, we resurrected our Frankenstein (a.k.a. ADACO) molecule by transfecting a mammalian expression construct into the human lung carcinoma A549 cell line. A stable cell line was selected by killing cells that did not contain the expression plasmid. In Figure 1A below, you can see the hypothetical structure of our molecule. We hypothesized that N- and C-terminal small chain fragment variable (scFV) antibody domains would fold independently from the Apaf1 CARD domain and that oppositely charged stretches of amino acids (blue and red lines) would separate and stabilize these domains. Figure 1B is a Western blot of normal A549 cells (---) and A549 cells that express the ADACO molecule. Antibodies targeting an internal His tag (red line) and the Apaf1 CARD domain confirmed expression of the ADACO. Interestingly, we did not detect the His tag via immunohistochemistry, suggesting that the native structure of ADACO precludes binding of the anti-His antibody. We interpret this as evidence of protein folding.

 

Excited with the high level of expression of our protein, we quickly challenged the cells with dengue virus (DENV) and then stained the cells using a kit that detects signs of cell death. Regulated cell death or apoptosis occurs when a stimulus induces the formation of an apoptosome. Clustering of our ADACO molecule should lead to the formation of an artificial apoptosome as the Apaf1 CARD domains oligomerize as shown in the video below:

Success! Figure 2 shows an increase in the number of Brdu+ cells when ADACO expressing cells are infected with DENV. DENV infection induces apoptosis in human cells that express ADACO.

 

Will ADACO expression protect human cell cultures from DENV infection?