Figure 1.
γ-Secretase activity in HeLa cells shows biphasic “activation-inhibition” dose-response curves in the presence of DAPT
[17], [18]. Modulation of γ-secretase activity by DAPT was measured by following secretion of Aβ 1–40 from HeLa cells using Aβ 1–40 specific ELISA. The observed biphasic dose-response curves can be described numerically using nonlinear regression and the equation 1 (Table 1). The calculated parameters allow tracing of two binding events, which can be separated mathematically as the activation phase and the inhibition phase (thin lines), with the corresponding EC50 and IC50 values (the vertical dashed lines). Different phases in the biphasic dose-response curve are marked with underlined numbers and the corresponding molecular interactions are illustrated schematically (C99 substrate can be shown as a transmembrane helix [75], while γ-secretase can be shown as a membrane embedded complex with a central aqueous cavity [76]). Complex 1 represents γ-secretase activity on its substrate in the absence of DAPT (31 pM of Aβ 1–40 secreted per 106 cells, Table 1). Complex 2; DAPT can activate γ-secretase activity on C99 substrate only if both DAPT and the substrate bind to γ-secretase simultaneously. Complex 3, complete saturation with DAPT leads to inhibition, indicating that there are at least two different binding events for DAPT, one activating and one inhibiting.
Table 1.
Best fit parameters for the biphasic activation-inhibition dose-response curves (eqn. 1)a.
Figure 2.
Gradual increase in the extent of γ-secretase saturation with its substrate leads to gradual changes from the biphasic to the standard dose-response curves.
Modulation of γ-secretase activity by DAPT was measured by following Aβ 1–40 secretion from HeLa cells using ELISA. The numbers next to each curve indicate pSG5-cDNAwtC99 in ng/mL, the profile at 0 ng/mL represents activity on the endogenous substrate (i.e. untransfected cells just as in Fig. 1). HeLa cells were transfected with increasing concentrations of pSG5-cDNAwtC99 plasmid to achieve gradual increase in expression of C99 substrate and Aβ 1–40 secretion (see methods). The observed profiles can be described numerically using equations 1 and 2, the best-fit values and the corresponding statistic are given in Table 2. (A) Different phases in the observed dose-response curves are marked by the underlined numbers to illustrate different molecular interactions schematically. The complexes 1, 2, and 3, represent different interactions between γ-secretase and DAPT at sub-saturating substrate as described in figure 1. A gradual increase in cDNAwtC99 results in a gradual increase in the enzyme activity at the lowest DAPT concentrations, and a decrease in the extent of enzyme activation by DAPT (Table 2). Thus, there is a direct competition between DAPT and the substrate for binding at the activation site (i.e. competition between complex 2 and 4). This indicates that there are at least two different binding sites for the substrate: the catalytic site and the site that can antagonize binding of DAPT at the activation sites (complex 4). (B) The peak activity is observed at around 250 ng/mL cDNAwtC99, at which point there is no more activation by DAPT, and only inhibition and standard dose-response curves can be observed (i.e. full transition from complex 1 to 4). Further increase in the substrate (i.e. cDNAwtC99 >250 ng/mL) leads to decrease in Aβ 1–40 secretion (Table 2B), which indicates that the substrate can also bind to the inhibition site (i.e. antagonism between complex 5 and complex 6).
Figure 3.
Biphasic inhibitors can reduce γ-secretase's capacity to process its substrates.
The molecular mechanism and the physiological significance of the biphasic activation-inhibition dose-response curves can be revealed by re-plotting the data from figures 2 A–B according to the standard approach for studies of modulators of enzyme activity ([33], or pp. 289–294 in ref. [31], or p. 251 in [34]). The Y-axis shows the reaction product, Aβ 1–40 secreted just as in Fig. 2. However the X-axis shows values that are functionally proportional to the extent of γ-secretase saturation with its C99 substrate; i.e. gradually increasing concentrations of cDNAwtC99 that was used to transfect the cells (as shown on the gel strip and described in the methods). The red line represents γ-secretase in the absence of DAPT at different level of saturation with its substrate, i.e. complex 1, 2 and 3. Aβ 1–40 secreted at 0 ng/mL cDNAwtC99 and 0 nM DAPT represents activity on the endogenous C99 substrate. Different curves represent different concentrations of DAPT in nM (i.e. activity modulator). Activation by DAPT can induce an apparent shift in the activity profiles to the left of the Y axis (symmetrically extrapolated dashed lines were used to illustrate the shift). This shift indicates that at sub-saturating substrate DAPT can activate γ-secretase by “filling-in” for the subsaturating substrate [31]. At the saturating substrate, DAPT is a noncompetitive inhibitor [31]. These synergistic activation-inhibition effects can drastically reduce γ-secretase's capacity to process it substrate (Table 3). Different sections on the graphs are labeled with the underlined numbers to map the corresponding interactions just as in the figures 1 and 2. Except for complexes 1, 3 and 4, this type of analysis cannot clearly resolve the activity range that corresponds to the different interactions as seen in figures 1 and 2.
Table 2.
Best fit parameters for the biphasic activation-inhibition dose-response curves with DAPTa.
Table 3.
Specified numerical values from the data shown in Fig. 3.a
Figure 4.
The biphasic dose-response curves (A) and the corresponding enzyme saturation profiles (B) can be modeled using numerical simulation.
The panels represent an attempt to simulate experimental data from Fig. 2 and Fig. 3, using the equation that can describe the proposed model mechanism (eqn. 3) and standard MS Excel program. In panel A the X axis shows inhibitor concentrations and different curves represent different substrate concentrations, the opposite combination is used to generate panel B. For both panels Vmax1 = 450, Vmax = 50, Kia = 15 nM, Kii = 120 nM, K0.5s = 300 nM, Ks2 = 500 nM.