PPP-IX binds to the protease domain of HtrA1 and disease-associated mutations eliminate binding.

Abstract

<p>(<b>A</b>) PPP-IX competed with HEMIN for endogenous HtrA1 binding. HeLa cell lysate was subjected to HEMIN pull down. Less HtrA1 was recovered in the presence of PPP-IX. Akt was used as a control and was not purified from the same pull down. (<b>B</b>) The HtrA1 protease domain was sufficient for interaction with MPPs. A schematic diagram of the deletion constructs tagged with HA and V5/hexa-His epitopes is shown (top). Cell lysates from HEK293 expressing variant HtrA1 proteins were subjected to HEMIN pull down and eluates (H) were examined for tagged protein with the anti-HA antibody (bottom left). The amount of recovered protein compared to input (I) is shown (bottom right). (<b>C</b>) PPP-IX competed with HEMIN for binding to the HtrA1 protease domain in a dose-dependent manner, with less HtrA1 protein recovery as PPP-IX concentration increased, revealed by immunoblotting for anti-HA. Akt was not similarly purified. The amount of recovered protein compared to input is shown (bottom). (<b>D</b>) Disease associated mutations reduced the interaction between HtrA1 and MPPs. The position of engineered single amino acid mutations in variant HtrA1 constructs is shown in the schematic diagram (top). Variant HtrA1 protein pulled down from transfected cell lysates with HEMIN agarose (H) was probed for HtrA1 (left panel) and the amount of recovered protein compared to input (I) was calculated (right panel). R274Q and V297 M significantly reduced the binding of HtrA1 to HEMIN (*: p<0.01). HtrA family member HtrA2 is not purified, demonstrating that the interaction is specific to HtrA1.</p