Interactions are specific and do not depend on overexpression

To further test the applicability of these synthetic domains as protein-interaction modules, the expression levels were analyzed. These domains might also interact with certain endogenous proteins (e.g., other DZF-containing proteins) and the observed activity and specificity might depend on overexpressing them. Thus, experiments were performed to rule out the possibility that the interactions of synthetic DZFs are dependent on overexpression. FLAG epitope-tagged versions of DZF-DBD and DZF-p65 fusion proteins were constructed, whose expression could be quantified by Western blotting using an anti-FLAG monoclonal antibody. To test, whether the interaction of the synthetic DZFs depends on their over-expression, the expression level of the DZF-p65 fusion proteins in VEGF-A activator reconstitution experiments was lowered by decreasing the amount of DZF-p65-encoding plasmid used to transfect the cells. Western blot analysis demonstrated that for three of the four synthetic DZF pairs, the level of p65-DZF was decreased by 7-fold or more without affecting DZF-DBD expression levels (Figure 3.11A). Although the amount of p65 was significantly reduced, these proteins were still able to mediate robust activation of VEGF-A (Figure 3.11B). In the case of the Ik-Ik-Hd and Pe-Pe-Eo DZFs, the level of Pe-Pe-Eo DZF-p65 fusion protein could not be decreased even when the amount of plasmid encoding this protein was reduced by 10-fold. Further reduction of the plasmid amount resulted in an impaired VEGF-A activation (data not shown). Thus, three of our four synthetic DZF pairs were able to mediate interaction even when the expression level of one member of the pair was reduced by >7-fold suggesting that overexpression of our synthetic DZF domains is not required to achieve interaction.

An additional set of experiments was performed to test, if the specificities of DZFs can be maintained even when they and their potential competitive interaction partners are both overexpressed. It was found that each of the four synthetic DBD-DZF hybrids interacts specifically with a DZF-p65 fusion harboring its synthetic interacting partner DZF but fails to interact with DZF-p65 fusions harboring either a synthetic non-interacting DZF or the wild-type TRPS1 DZF (Figure 3.12A). Importantly, Western blot analysis of cells expressing these proteins revealed that within each set of experiments each DZF-DBD fusion protein and its

3 2

1 4 5 6 7 8

relative level of p65 hybrid expression

fold-decrease in p65 hybrid expression 7.0X 9.5X 1.7X 10.5X

2.1 0.31.9 0.2 1.50.9 2.10.2

Fold-activation of VEGF-A protein expression Tr-Eo-Eo + Eo-Eo-Hd (hi)

Ik-Ik-Hd + Pe-Pe-Eo (hi)

Pe-Pe-Eo + Eo-Eo-Hd (hi) DBD hybrid p65 hybrid

Hl-Eo-Eo + Pe-Hd-Hd (hi) Tr-Eo-Eo + Eo-Eo-Hd (low)

Hl-Eo-Eo + Pe-Hd-Hd (low)

Ik-Ik-Hd + Pe-Pe-Eo (low)

Pe-Pe-Eo + Eo-Eo-Hd (low)

B

3 2

1 2 3 4 5 6 7 8

1 4 5 6 7 8

relative level of p65 hybrid expression

fold-decrease in p65 hybrid expression 7.0X 9.5X 1.7X 10.5X7.0X 9.5X 1.7X 10.5X

2.1 0.31.9 0.2 1.50.9 2.10.2

Fold-activation of VEGF-A protein expression Tr-Eo-Eo + Eo-Eo-Hd (hi)

Ik-Ik-Hd + Pe-Pe-Eo (hi)

Pe-Pe-Eo + Eo-Eo-Hd (hi) DBD hybrid p65 hybrid

Hl-Eo-Eo + Pe-Hd-Hd (hi) Tr-Eo-Eo + Eo-Eo-Hd (low)

Hl-Eo-Eo + Pe-Hd-Hd (low)

Ik-Ik-Hd + Pe-Pe-Eo (low)

Pe-Pe-Eo + Eo-Eo-Hd (low) Tr-Eo-Eo + Eo-Eo-Hd (hi)

Ik-Ik-Hd + Pe-Pe-Eo (hi)

Pe-Pe-Eo + Eo-Eo-Hd (hi) DBD hybrid p65 hybrid

Hl-Eo-Eo + Pe-Hd-Hd (hi) Tr-Eo-Eo + Eo-Eo-Hd (low)

Hl-Eo-Eo + Pe-Hd-Hd (low)

Ik-Ik-Hd + Pe-Pe-Eo (low)

Pe-Pe-Eo + Eo-Eo-Hd (low)

B

Figure 3.11 Synthetic DZF-DZF interactions do not critically depend upon protein over-expression. (A) Western blot analysis of human cells transfected with various amounts of DZF-encoding plasmids. Western blots were performed on 293 cells transfected with combinations of FLAG-tagged DBD-DZF and p65-DZF hybrid proteins using an anti-FLAG monoclonal antibody. For each pair of DZF hybrid, two different amounts of DZF-p65-encoding plasmid were used (“hi” = 0.25 ug DNA,

“low” = 0.06 ug DNA). The amount of cell lysate loaded in each lane was normalized to the number of viable cells determined by WST1 assay (see section 2.3.2.3). Band intensities were quantified using BioRad Quantity One software and a BioRad Fluor-S MultiImager instrument. The relative intensities of each DZF-p65 band are shown above each lane and represent the average of band intensities from two assays. Relative values were calculated by normalizing the intensity of each DZF-p65 band to the associated DZF-DBD band. (B) VEGF-A assays were performed simultaneously on culture supernatants of cells used for the Western blot analysis. Fold-stimulation of VEGF-A protein expression was calculated by measuring the VEGF-A contents in the culture medium using ELISA. Bars shown represent mean fold-activations of VEGF-A expression determined from two independent experiments and error bars indicate standard errors of the mean. Identities of subdomains in the synthetic DZFs are abbreviated as in Figure 3.4. This Figure was taken from Giesecke et al. (2006).

three corresponding p65-DZF fusions (harboring an interacting synthetic DZF, a non-interacting synthetic DZF and the DZF from wild-type TRPS1) were expressed at similar levels (Figure 3.12B). This suggested that the specificities of DZF interactions are maintained even when the DZF hybrid proteins are overexpressed. Furthermore, since TRPS1 is widely expressed in many different human cell types (Momeni et al., 2000) these results also suggest that most of the synthetic DZFs do not effectively interact with potential endogenous competitors, even when these are very abundant.

0 5 10 15 20

1 DBD hybrid p65 hybrid

Tr-Eo-Eo + Eo-Eo-Hd

Fold-activation of VEGF-A protein expression

3 DBD hybrid p65 hybrid

Tr-Eo-Eo + Eo-Eo-Hd

Fold-activation of VEGF-A protein expression

3

Figure 3.12 Overexpression of non-interacting synthetic DZFs does not result in an activation of VEGF-A. (A) Expression of VEGF-A from human 293 cells transfected with plasmids expressing various combinations of interacting and non-interacting DBD-DZF and p65-DZF hybrid proteins.

Fold-stimulation of VEGF-A protein expression was calculated as described in Figure 3.11. Identities of subdomains present in the synthetic DZFs are abbreviated as in Figure 3.4. (B) Western blots of whole cell lysates prepared from the transfected cells used for ELISA assays in (A) are shown. Blots were performed as described in Figure 3.11. This Figure was taken from Giesecke et al., (2006).