Dataset Information

Regulation of the HTRA2 protease activity by PDZ domain-binding peptide ligands and the influence on HTRA2-specific protein interaction networks in retinal tissues

ABSTRACT: For the identification of HTRA2-specific protein interaction partners in retinal tissues of the house swine (Sus scrofa), we performed co-immunoprecipitation experiments with recombinant 6His-tag HTRA2 followed by quantitative LC-MS-based proteomics (see Fig. 2A and File S1). In brief, 2 µg recombinant 6His-tag HTRA2 (HTRA2 group) were spiked into 5 mg homogenized porcine retina and was subsequently enriched with the bounded protein interaction partners via Ni-NTA affinity purification (n=3). In addition, two further sample groups were treated with 10 µg UCF 101 (UCF-101 group) or 10 µg synthetic CDR peptide (CDR group) to unravel the influence of both molecules on the HTRA2-specific protein interactome in the retinal tissues (n=3 for each group). Eluate fractions of each group were separated by 1-D SDS PAGE (Fig. S1) and subsequently subjected to in-gel trypsin digestion. To distinguish unspecific from HTRA2 specific binders, we also included a control Ni-NTA bead group (CTRL group) for the quantitative MS analysis (n=3).

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Retina

DISEASE(S): Open-angle Glaucoma

SUBMITTER: Carsten Schmelter

LAB HEAD: Dr. Carsten Schmelter

PROVIDER: PXD024442 | Pride | 2021-10-11

REPOSITORIES: Pride

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Publications

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.

Schmelter Carsten C Fomo Kristian Nzogang KN Perumal Natarajan N Pfeiffer Norbert N Grus Franz H FH

Biomedicines 20210813 8

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, e ...[more]

PMID: 34440217

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Project description:Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A’s N terminal LRR domain.

Dataset Information

Regulation of the HTRA2 protease activity by PDZ domain-binding peptide ligands and the influence on HTRA2-specific protein interaction networks in retinal tissues

Publications

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.

Similar Datasets

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