Characterization of various double-DZFs

To initially test whether double-DZFs could mediate specific interactions, the mammalian cell-based activator reconstitution assay was used. As shown in Figure 3.16, only double-DZF1/double-DZF5 and double-DZF4/double-DZF7 pairs were able to mediate robust activation of VEGF-A expression.

By contrast, testing different combinations of the same double-DZFs (double-DZF1/double-DZF7 and double-DZF4/double-DZF5) resulted in a lower VEGF-A expression level similar to the one obtained with single DZFs. Interestingly, these pairs of double-DZFs only differed in the linear order of their constituent synthetic DZFs. The remaining double-DZFs exhibited a lower level of VEGF-A activation. Surprisingly, the only difference between some of these double-DZFs is the linker region between the single DZFs pairs. For example double-DZF1 and double-DZF5 mediated efficient activation of VEGF-A expression. The single DZFs in these two DZFs are linked by the amino acids GEKP. In DZF2 and double-DZF6 the same single DZFs were fused in the same linear order using the hinge linker and the activation of VEGF-A mediated by these two double-DZFs was less efficient. A simple explanation of these results is that the linker may play a role in orientating the two single DZFs in the double-DZFs and is therefore important for the ability of the double-DZFs to interact with another double-DZF. It can certainly be ruled out that the linker is part of the

Figure 3.16 Synthetic double-DZFs interact in mammalian cells. Pairwise combinations of plasmids encoding double-DZFs (dDZF) were co-transformed into HEK293 cells and interactions of these pairs were assessed using the mammalian cell-based activator reconstitution assay. VEGF-A protein expression levels were calculated as absolute values using ELISA measurements of secreted VEGF-A that was performed two times. Identities of dDZF domains as indicated in Figure 4.15C.

interacting surface because both double-DZF1/double-DZF7 and double-DZF4/double-DZF5 pairs were not able to mediate a robust activation of VEGF-A, although they harbor the same linker region as double-DZF1/double-DZF5 and double-DZF4/double-DZF7 pairs which were able to active VEGF-A very efficiently.

We were interested in comparing the interaction strength of the double-DZFs with the level of interaction observed with the single DZF pairs that we used to construct these double-DZFs. As shown in Figure 3.17, both double-DZF pairs (double-DZF1/double-DZF5 and double-DZF4/double-DZF7) mediated very robust activation of VEGF-A expression, greater than the activation observed with the single DZF pairs from which they were constructed.

The level of activation achieved by the double-DZFs is similar to that obtained with the originally described artificial VEGF-A activator (SpI-p65 in Figure 3.17; Liu et al., 2001).

Control experiments demonstrated that the expression of neither the DBD-double-DZF protein nor the p65- double-DZF protein alone activated VEGF-A expression. Interactions of

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Eo-Eo-Hd + double-DZF 5 Hl-Eo-Eo + double-DZF 5 ---- + double-DZF 5

Eo-Eo-Hd + double-DZF 7 Hl-Eo-Eo + double-DZF 7 ---- + double-DZF 7

Fold-activation of VEGF-A protein expression

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Eo-Eo-Hd + double-DZF 5 Hl-Eo-Eo + double-DZF 5 ---- + double-DZF 5

Eo-Eo-Hd + double-DZF 7 Hl-Eo-Eo + double-DZF 7 ---- + double-DZF 7

Fold-activation of VEGF-A protein expression

Figure 3.17 Analysis of the interactions mediated by double-DZFs in mammalian cells. The strength and mode of interactions mediated by the double-DZF1/double-DZF5 and double-DZF4/double-DZF7 pairs was assessed using the mammalian cell-based activator reconstitution assay. dDZFs were tested for their interaction with each other and with the single DZFs from which they were derived. Controls testing the interaction of single DZF pairs and of the intact synthetic VEGF-A activator SpI-p65 (consisting of the DBD that binds the VEGF-A promoter covalently fused to the p65 activation domain) are also shown.

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 calculated from three independent assays and error bars indicate standard errors of the mean. Identities of the synthetic DZFs are abbreviated as in Figure 3.4, and identities of dDZF domains are as indicated in Figure 3.15C.

the double-DZFs with the individual single DZFs which they consist of caused a reduced activation of VEGF-A compared to the activation obtained with the double-DZFs. In addition, when the linear order of the synthetic DZFs was reversed, both double-DZF pairs (double-DZF1/double-DZF7 and double-DZF4/double-DZF5) stimulated VEGF-A expression less efficiently. This suggests that 1/5 and double-DZF-4/double-DZF-7 interact stronger because they are able to utilize both single DZFs whereas double-DZF-1/double-DZF-7 and double-DZF-4/double-DZF-5 interact less strongly because they use only one of the two DZFs for mediating the interaction.

To further confirm this, another set of double-DZFs was designed where each of the single DZFs in every double-DZF construct was individually mutated by introducing the D18Q mutation. A double-DZF construct that harbors such a mutation in one of its DZFs was then tested for its ability to interact with another double-DZF construct, which harbors a mutation in the corresponding interacting DZF domain (i.e., two single DZF domains that usually interact with each other). Both of the double-DZF pairs were impaired in their ability to activated VEGF-A expression when they harbored mutations in interacting single DZFs. The activation level was similar to that observed with the single DZF pairs suggesting that only the wild-type DZFs in the double-DZF constructs mediated the interaction (Figure 3.18). This confirms that the two wild-type double-DZF pairs are able to mediate a very robust activation

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1 DBD-fusion p65-fusion

double-DZF 4 + double-DZF 7

Tr-Eo-Eo + Eo-Eo-Hd Hl-Eo-Eo + Pe-Hd-Hd double-DZF 1 + double-DZF 5 double-DZF 1* + double-DZF 5*

double-DZF 1** + double-DZF 5**

double-DZF 4* + double-DZF 7*

double-DZF 4** + double-DZF 7**

Fold-activation of VEGF-A protein expression

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1 DBD-fusion p65-fusion

double-DZF 4 + double-DZF 7

Tr-Eo-Eo + Eo-Eo-Hd Hl-Eo-Eo + Pe-Hd-Hd double-DZF 1 + double-DZF 5 double-DZF 1* + double-DZF 5*

double-DZF 1** + double-DZF 5**

double-DZF 4* + double-DZF 7*

double-DZF 4** + double-DZF 7**

Fold-activation of VEGF-A protein expression

Figure 3.18 Analysis of the effect of the D18Q mutation introduced into the single DZFs present in the double-DZFs. The D18Q mutation was introduced into one of the single DZFs present in the DBD-dDZF hybrid and into the corresponding single DZF present in the p65-dDZF hybrid. One star (*) indicates that the mutation was introduced into the single DZFs Hl-Eo-Eo and Pe-Hd-Hd present in each of the dDZF domains, while two stars (**) indicate that the mutation was introduced into the Tr-Eo-Eo / Tr-Eo-Eo-Hd DZF domains. Note that all dDZFs consist of combinations and different arrangements of these single DZFs (see Figure 3.15C). Controls testing the interaction of the intact dDZFs and the single DZF pairs are also shown. Fold-stimulation of VEGF-A protein expression was calculated from three independent assays and error bars indicate standard errors of the mean.

of VEGF-A by using both single DZFs for the interaction, thus displaying an extended interaction surface.