Regulation of the HTRA2 Protease Activity by an Inhibitory Antibody-Derived Peptide Ligand and the Influence on HTRA2-Specific Protein Interaction Networks in Retinal Tissues

Abstract

The mitochondrial serine protease HTRA2 has many versatile biological functions ranging from being an important regulator of apoptosis to being an essential component for neuronal cell survival and mitochondrial homeostasis. Loss of HTRA2 protease function is known to cause neurodegeneration, whereas overactivation of its proteolytic function is associated with cell death and inflammation. In accordance with this, our group verified in a recent study that the synthetic peptide ASGYTFTNYGLSWVR, encoding the hypervariable sequence part of an antibody, showed a high affinity for the target protein HTRA2 and triggered neuroprotection in an in vitro organ culture model for glaucoma. To unravel this neuroprotective mechanism, the present study showed for the first time that the synthetic CDR1 peptide significantly (p < 0.01) inhibited the proteolytic activity of HTRA2 up to 50% using a specific protease function assay. Furthermore, using state-of-the-art co-immunoprecipitation technologies in combination with highresolution MS, we identified 50 significant protein interaction partners of HTRA2 in the retina of house swine (p < 0.01; log2 fold change > 1.5). Interestingly, 72% of the HTRA2-specific interactions (23 of 31 binders) were inhibited by additional treatment with UCF-101 (HTRA2 protease inhibitor) or the synthetic CDR peptide. On the other hand, the remaining 19 binders of HTRA2 were exclusively identified in the UCF101 and/or CDR group. However, many of the interactors were involved in the ER to Golgi anterograde transport (e.g., AP3D1), aggrephagy (e.g., PSMC1), and the pyruvate metabolism/citric acid cycle (e.g., SHMT2), and illustrated the complex protein interaction networks of HTRA2 in neurological tissues. In conclusion, the present study provides, for the first time, a comprehensive protein catalogue of HTRA2-specific interaction partners in the retina, and will serve as reference map in the future for studies focusing on HTRA2mediated neurodegeneration.

Contribution of the Commensal Microflora to the Immunological Homeostasis and the Importance of Immune-Related Drug Development for Clinical Applications

“…The newest findings of our group verified the inhibitory effect of synthetic CDR1 on the protease function of HTRA2 and is most likely caused by physical interaction with the PDZ domain [147]. Furthermore, HTRA2 specifically interacts with a wide range of proteins in the retina comprising important regulatory pathway mechanism ER to Golgi anterograde transport (e.g., AP3D1), protein localization to membrane (e.g., FLNA), aggrephagy (e.g., PSMC1) and the pyruvate metabolism/citric acid cycle (e.g., SHMT2) [147].…”

“…The newest findings of our group verified the inhibitory effect of synthetic CDR1 on the protease function of HTRA2 and is most likely caused by physical interaction with the PDZ domain [147]. Furthermore, HTRA2 specifically interacts with a wide range of proteins in the retina comprising important regulatory pathway mechanism ER to Golgi anterograde transport (e.g., AP3D1), protein localization to membrane (e.g., FLNA), aggrephagy (e.g., PSMC1) and the pyruvate metabolism/citric acid cycle (e.g., SHMT2) [147]. Thereby, the exogenous application of synthetic CDR1 peptide or protease inhibitor UCF-101 (blocking the catalytic domain of HTRA2) highly influenced the HTRA2specific interactome in the retina and might highlight the importance of these signaling pathways in the pathogenesis of glaucoma.…”

Inhibition of High-Temperature Requirement Protein A2 Protease Activity Represses Myogenic Differentiation via UPRmt

“…To test this hypothesis, we treated murine C2C12 myoblasts with UCF101, a specific inhibitor of HtrA2, and performed myogenic differentiation. The molecular structure of UCF101 is shown in Figure 2 a; in previous studies, it has been reported to have a significant inhibitory effect on HtrA2 protease activity at a concentration of 20 μM [ 36 , 37 , 38 ]. C2C12 myoblasts were treated with different concentrations of UCF101 for 48 h. The results showed that cell viability was not affected when the concentration of UCF101 was below 20 μM ( Figure 2 b).…”

“…We found a repressive effect of UCF101 on C2C12 myogenic differentiation both microscopically and at the molecular level. UCF101 has been used in numerous studies for its ability to significantly inhibit the protease activity of HtrA2, thus preventing the binding of HtrA2 to proteins involved in unfolded protein reactions and pyruvate metabolism in mitochondria [ 36 , 37 , 41 ]. In our experiments, we found no significant change in the level of apoptosis in the UCF101 treated and untreated groups.…”

“…C2C12 myoblasts were seeded into 24-well plates and treated with UCF101 or were left untreated. It has been suggested through many in vitro and in vivo studies that UCF-101 can specifically inhibit HtrA2 protease activity at a concentration of 20 μM [ 36 , 37 , 38 ]. On D3 and D5, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 7 min.…”