Additional file 1 of Intracellular delivery of Parkin-RING0-based fragments corrects Parkin-induced mitochondrial dysfunction through interaction with SLP-2
Creators: name: Zanon, Alessandra
name: Guida, Marianna
name: Lavdas, Alexandros A.
name: Corti, Corrado
name: Castelo Rueda, Maria Paulina
name: Negro, Alessandro
name: Pramstaller, Peter P.
name: Domingues, Francisco S.
name: Hicks, Andrew A.
name: Pichler, Irene
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Cancer
subject: Virology

description: Additional file 1: Table S1. Primer sequences used to amplify the Parkin domains. Table S2. Genotypic and phenotypic information for investigated PD patients with PRKN mutations. Table S3. Predicted candidate binding sites in Parkin RING0 that overlap with PD-causing mutations. Figure S1. The interaction between Parkin and SLP-2 is detected by using the Tripartite split GFP assay in E. coli. Figure S2. The transfected RING0 domain localises partially to the mitochondria. A) HeLa cells were transfected with RING0 with mitochondrial targeting sequence (green: anti-myc; red: anti-GRP75; blue: nuclei) or B) with RING0-HA (green: anti-HA; red: anti-GRP75; blue: nuclei). The transfected RING0 domain localizes partially to the mitochondria, whereas the mitochondrial targeting sequence induces complete translocation of RING0 to the mitochondria. Scale bar: 25 µm. Figure S3. Overexpressed RING0 domain and its mutants in HeLa cells are present at the mitochondria. Wildtype and mutant RING0 domains (carrying either a P153A, K161A, or K211A mutation) are present at the mitochondria (green) and to a very low extend at the lysosomes (blue). green: GRP75; blue: GFP-LC3b; red: HA-RING0 and HA-RING0 mutants; white: nuclei. Scale bar: 10 µm. Figure S4. Oxygen consumption impairment in Parkin-deficient cells improves after Parkin mini-peptide application. (A) Representative curves of fluorescence signal, generated by the oxygen probe, reflecting dissolved oxygen in the culture medium of WT SH-SY5Y, Parkin KD SH-SY5Y, and Parkin KD SH-SY5Y cells treated with Parkin mini-peptide #1. (B) Relative quantification of oxygen consumption. Cellular respiration is significantly increased by mini-peptide #1. Error bars represent the mean ± SEM of 3 data points. Statistical differences were determined using one-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ns = not significant. RFU: relative fluorescence units. Figure S5. Mitochondrial ROS levels in Parkin-deficient cells after Parkin mini-peptide application for 3 h and 8 h, respectively. Delivery of mini-peptide-conjugates #1 (iii), #3 (v), #4 (vii) are not able to reduce mitochondrial ROS production in Parkin KD SH-SY5Y cells (ii) after application for 3 h. (i) WT SH-SY5Y cells. After 8 h of peptide application, a significant effect is detected for mini-peptide-conjugates #3 (vi) and #4 (viii). Statistical differences were calculated by one-way ANOVA followed by Holm-Sidak post hoc test to correct for multiple comparisons. ** p ≤ 0.01, **** p ≤ 0.0001. Scale bar: 50 μm. Figure S6. Detection of Parkin mini-peptide marked with a GFP-tag. SH-SY5Y cells were treated with Parkin mini-peptide (#1) and mitochondria stained with MitoTracker red fluorescent dye. Live cell imaging detected the Parkin mini-peptide in SH-SY5Y cells 3 h (A) and 16 h after application (B), respectively. red: MitoTracker red; green: GFP-Parkin mini-peptide #1. Scale bar: 50 µm. Figure S7. Parkin mini-peptide conjugates rescue altered mitochondrial membrane potential levels (Δψ) in hiPSC-derived neurons carrying endogenous PRKN mutations. Mitochondrial membrane potential is increased by mini-peptides #1 (C), #3 (D), and #4 (E) in hiPSC-derived neurons carrying PRKN mutations (PRKN NT, not treated, B). hiPSC-derived neurons of control individuals (A). Scale bar: 20 μm. Statistical differences were calculated by one-way ANOVA followed by Holm-Sidak post hoc test to correct for multiple comparisons **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.