MPPs and HtrA1 appear to interact directly.

<p>(<b>A</b>) MPPs displayed differential activity in affecting The pattern of extracellular HtrA1 complexes (arrowheads) from HeLa cell low-serum conditioned medium is differentially altered by treatment with MPPs (25 mM, 37°C, 1 hr), as demonstrated when probed with polyclonal anti-HtrA1 antibody. (<b>B</b>) MPPs may induce conformational changes upon binding to HtrA1. The accessibility of N-terminal and C-terminal epitopes in the presence of MPPs was determined by ELISA and compared to DMSO controls. Error bars indicate the standard deviation. (<b>C</b>) Extracellular HtrA1 (arrows) could be precipitated from HEK-HtrA1 conditioned medium using HEMIN-conjugated agarose beads but not control, unconjugated agarose beads. (<b>D</b>) Competitive binding experiments using conditioned medium from HEK-HtrA1 or HeLa cells pre-incubated with MPPs revealed reduced recovery of HtrA1 (arrows) in the presence of competitor compounds when probed with polyclonal anti-HtrA1 antibody. (<b>E</b>) Degradation of Fibulin 5 in fixed HeLa cells treated with HtrA1 conditioned medium was enhanced in the presence of TPP, ZPP and PPP-IX. RMA: Rosmarinic acid, TPP: tin protoporphyrin IX, ZPP: zinc protoporphyrin IX, PPP-IX: protoporphyrin IX, HEM: HEMIN, CPP: cobalt protoporphyrin IX, CM: conditioned medium.</p

The formation of HtrA1 extracellular complexes can be affected by small molecules.

<p>(<b>A</b>) A schematic of the chemical intervention strategy for selecting molecules capable of affecting extracellular HtrA1 complex formation. “Hit compounds” were identified when the HtrA1 complex pattern was altered in comparison to DMSO treatment. (<b>B</b>) Conditioned serum from HEK-HtrA1 and HeLa cells was probed with anti-HA and anti-HtrA1 antibody respectively after treatment with the seven hit compounds. All compounds increased HtrA1 complex abundance (arrowheads) compared to the DMSO treated control. DMSO treatment did not alter complex formation. CBD: (S)-(-)-Carbidopa, RMA: Rosmarinic acid, ZPP: zinc protoporphyrin IX, TPP: tin protoporphyrin IX, ACTD: Actinomycin D, YM: YM 90709, AZ: AZ 10417808. (<b>C</b>) Schematic diagrams of the chemical structure of the non-MPP hit compounds. Dashed lines indicate a conserved chemical moiety in Carbidopa and Rosmarinic acid. (<b>D</b>) Schematic diagram of the chemical structures of the protoporphyrin IX-based metalloporphyrins used.</p

Formation of oligomer complexes by secreted HtrA1 protein in cell culture.

<p>(<b>A</b>) Serum rich (10%) conditioned medium from HeLa cells contained a higher level of HtrA1 complexes than low serum (0.2%) conditioned medium. Medium was collected at the indicated time points and subjected to non-reducing (- β-ME) or reducing (+ β-ME) SDS-PAGE. Immunoblotting with polyclonal anti-HtrA1 antibody detected full length HtrA1 (arrow) and HtrA1-containing complexes (arrowheads) under non-reducing conditions. Full length HtrA1 could be detected under non-reducing conditions. (<b>B</b>) Serum rich and low serum conditioned media subjected to native PAGE revealed two prominent bands when probed with monoclonal anti-HtrA1 antibody (bracket). A dilution series is shown for clarity. (<b>C</b>) A schematic diagram of the human HtrA1 expressed from the full-length construct, tagged with an HA epitope (red) at the N-terminus and V5/hexa-His tag (blue) at the C-terminus. S denotes the signal peptide. (<b>D</b>) Exogenous HtrA1 is stably expressed in HEK293 cells. HtrA1 could be detected in conditioned medium from HeLa cells and stably transfected HEK-HtrA1 cells, but not that from HEK293 cells or medium that has not been exposed to cells, when probed with monoclonal anti-HtrA1 antibody (middle panel). Probing with anti-V5 antibody (right panel) detected specific bands (arrows, arrowheads) only in HEK-HtrA1 conditioned medium, although a non-specific band was detected in all samples (asterisk). Coomassie blue staining is included as a loading control (left panel). (<b>E</b>) Serum rich (10%) conditioned medium contained a higher level of exogenous HtrA1 complexes than serum free conditioned medium. Exogenous HtrA1 was captured from conditioned medium via the hexa-His tag, using Ni-NTA columns. Full-length HtrA1 (arrow) could be detected in the input conditioned medium (control) and column-captured fraction (bound) under both serum conditions. HtrA1 complexes (arrowheads) were only detected under serum rich conditions. CM: conditioned medium.</p

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

<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