Intracellular delivery of Parkin-RING0-based fragments corrects Parkin-induced mitochondrial dysfunction through interaction with SLP-2

• Published in: Journal of translational medicine Vol. 22; no. 1; pp. 59 - 18
• Main Authors: Zanon, Alessandra, Guida, Marianna, Lavdas, Alexandros A, Corti, Corrado, Castelo Rueda, Maria Paulina, Negro, Alessandro, Pramstaller, Peter P, Domingues, Francisco S, Hicks, Andrew A, Pichler, Irene
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 16-01-2024; BioMed Central; BMC
• Subjects: Analysis; Biosynthesis; Care and treatment; Computational neuroscience; Crystal structure; Diagnosis; Dopamine; Electron transport; Fibroblasts; Genes; Genetic aspects; Immunofluorescence; Insects; Missense mutation; Mitochondria; Mitochondrial DNA; Movement disorders; Mutation; Neuroblastoma; Neuroblasts; Neurodegenerative diseases; Neurons; Neurophysiology; Parkin; Parkin mini-peptide; Parkin protein; Parkinson's disease; Peptides; Phenotypes; Pluripotency; Protein structure; Protein-protein interactions; Proteins; Recovery of function; SLP-2; Germany; SLP-2; Mitochondria; Parkinson′s disease; Parkin mini-peptide; Parkin
• ISSN: 1479-5876; 1479-5876
• DOI: 10.1186/s12967-024-04850-3

Loss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson's disease (PD). We have previously identified mitoch ondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function.
In fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function.
Using a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations.
These findings place further emphasis on the importance of the protein-protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein-protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.