Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 1 PARL over-expression leads to increased processing of Pink1-66. (a) Schematic representation of human Pink1. The predicted matrix targeting sequence (MTS), the transmembrane domain (TMD), the kinase domain and the putative PARL processing site are indicated. Comparison of the TMDs of human (Hs) and D. melanog- aster (Dm) Pink1 using the EMBOSS pair- wise alignment algorithm reveals significant sequence conservation. The hydrophobicity plot of the relevant region is shown [using the scale of Kyte and Doolittle (1982), with a window size of 7] indicating the potential TMD boundaries. (b) Co-expression of hu- man Pink1 with PARL leads to an increased processing of the full-length 66 kDa form (open triangle). A catalytically inactive PARL mutant (SA) shows no activity. Subcellular fractionation reveals that Pink1-66 and the processed Pink-55 (filled triangle) are found in mitochondria (m) and also the non-mito- chondrial soluble fraction (c). IRES GFP and the cellular markers VDAC, AIF and actin were used as transfection and loading con- trol. t, total cell extract (10% loaded). (c) Quantification of Pink1 66/55 kDa ratio in the mitochondrial fraction upon PARL wt and SA over-expression (n = 3, means ± SEM). Significant changes versus mock are indi- cated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). (d) Titration of PARL cDNA significantly increases processing of Pink1-66 (n = 3, means ± SEM). Significant changes versus mock are indicated (*p < 0.05; One-way ANOVA with Bonferroni’s post-test).

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Over Expression, Sequencing, Comparison, Expressing, Mutagenesis, Activity Assay, Fractionation, Transfection, Titration

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 2 PARL is required for Pink1 processing. (a) Left panel: PARL knockdown by two non-overlapping siRNAs (PARL-1 and -2) blocks Pink1-66 processing and reduces the level of Pink1-55, when com- pared to a non-targeting siRNA (nt). Other proteins like VDAC, AIF, actin and IRES GFP are not affected. t, total cell extracts; m, mito- chondrial pellet and c, non-mitochondrial fraction. Right panel: Quan- tification of the Pink1 66/55 kDa ratio in the mitochondrial fractions upon knockdown of endogenous PARL by siRNAs (means ± SEM, n = 3). Significant changes versus mock are indicated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). Right bottom panel: PARL knockdown was monitored in the mitochondrial fraction. (b) Left panel: Doxycyclin-induced expression of PARL-specific shRNA in Hek293- shRNA cells for 6 days diminishes Pink1-66 processing. For PARL

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Knockdown, Expressing, shRNA

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 4 Both uncoupling of the mitochondrial membrane potential and PARL ablation lead to the accumulation of Pink1-66 at the outer mitochondrial membrane. (a) Upper panel: Uncoupling of the mitochondrial membrane potential in Hek293-shRNA cells (by CCCP) leads to a Pink1 66/55 kDa ratio as observed upon PARL knockdown (shRNA). Total cell extracts (t), mitochondrial fractions (m) and non-mitochondrial soluble fractions (c) were analyzed. Lower panel: Quantification of Pink1 66/55 kDa ratio of the indicated fractions is shown (means ± SEM, n = 3). Mitochondrial Pink1 66/ 55 kDa ratio is significantly lower after PARL shRNA and CCCP treatment than after treatment either with PARL shRNA or CCCP alone (*p < 0.05; **p < 0.01; paired t-test). (b) Upper panel: Outline of the Pink1 construct with a juxtamembrane factor Xa protease cleavage site (Pink1Xa). Lower panel: Mitochondria isolated from cells expressing Pink1Xa were subjected to digestion by factor Xa protease. Factor Xa cleaves Pink1-66 (open triangle) but not Pink1-55 (filled triangle) generating a 52 kDa form (grey triangle). In mitochondria isolated from cells expressing the PARL-specific shRNA or CCCP-treated cells, the factor Xa-sensitive outer membrane form of Pink1-66 increased. Note that upon PARL knockdown Pink1Xa was cleaved by an alternative protease within the N-terminal MTS leading to a 64 kDa form (labeled with an asterisk), which is not observed upon expression of Pink1 wild type.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Membrane, shRNA, Knockdown, Construct, Isolation, Expressing, Labeling

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 5 Pink1 TMD mutations block PARL-catalyzed processing. (a) TMD sequences of Pink1 wild type (wt), Pink1G107L, Pink1G109L, Pink1R98W and Pink1I111S are shown. Mutated residues are highlighted in bold. (b) and (c) Western analysis of Pink1 mutants expressed in Hek293-shRNA cells after doxycyclin-induced knockdown of PARL (shRNA) and CCCP treatment (total cell extracts are shown). Notably, for Pink1G107L, Pink1G109L and Pink1R98W, the Pink1-66 precursor was modified by an unknown post-translational modification leading to a 68 kDa species (labeled by an asterisk). Quantification of the Pink1 66/55 kDa ratio in the indicated fractions is shown (means ± SEM, n = 3). Pink1-55 levels are lower for all probed mutants tested. Pink1G107L and Pink1R98W processing is insensitive to PARL ablation showing highly reduced processing of these mutants by PARL.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Blocking Assay, Western Blot, shRNA, Knockdown, Labeling

Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Gene and protein expression analysis for (a and b) PINK1 and (c and d) Parkin. For gene expression analysis [men (M): n = 8; women (W): n = 8], data were normalized to Men ECC + REST at BL. A time × sex × condition interaction was observed for PINK1 . For protein expression analysis [M: n = 6; W: n = 6], data were normalized to Men ECC + REST at BL. A sex × condition × time interaction was observed for Parkin. A time × sex interaction was observed for Parkin. A condition × sex interaction was observed for Parkin. The box and whisker plots display an example of BL, 12 h, and 24 h loading gene and protein expressions for PINK1 and Parkin (see Figure 2b for the corresponding GAPDH). MW, molecular weight (kDa). * p < 0.05 versus ECC + REST. φ P < 0.05 versus BL. $ p < 0.05 versus ECC+REST. γ P< 0.05 versus BL. # p < 0.05 versus BL

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing, Whisker Assay, Molecular Weight

Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Bivariate correlational analysis for gene expression of (a) DRP1 , (b) PINK1 and the protein expression of (c) OPA1, (d) PINK1, and (e) Parkin with testosterone area under the curve. The average of 12 and 24 h were used for gene and protein expression in correlational analyses

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing

Journal: Physiological Reports

Article Title: Sex‐specific mitochondrial dynamics and mitophagy response to muscle damage

doi: 10.14814/phy2.15230

Figure Lengend Snippet: Bivariate correlational analysis for gene expression of (a) MFN1 , (b) DRP1 , and protein expression of (c) Parkin with the cortisol area under the curve (AUC) and gene expression of (d) PINK1 with growth hormone AUC. The average of 12 and 24 h were used for gene and protein expression in correlational analyses

Article Snippet: Samples were then transferred electrophoretically to a PVDF membrane at 70 V at 4°C for 2.5 h. Next, 5% non‐fat milk powder was used to block the membranes for 1 h, and after that, the membrane was incubated with primary antibodies against OPA1 (1:1000; Cell Signaling, 80471S; 80 kDa), DRP1 (1:1000; Novus, NB110‐55288; 80 kDa), PINK1 (1:2000; Novus, NBP1‐49678; 55 kDa), Parkin (1:1000; Cell Signaling, 2132S; 55 kDa), and GAPDH (1:5000; Santa Cruz, sc‐365062; 37 Membranes were stripped) with 3% non‐fat milk at 4°C overnight.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: Mitofusin degradation requires PINK1 expression. (a) M17 cells expressing control or PINK1 shRNA were treated with CCCP or CCCP plus MG132. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 20 µM for 4 h; MG132, 30 µM, 30 min prior and with CCCP. (b) The average of Mfn1 or Mfn2 levels normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from panel a. The protein levels in control (nontreated, control shRNA) cells were set as 1. (c) HeLa cells stably transfected with YFP-Parkin were pretreated with CHX before treatment with CCCP, as indicated on top. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 10 µM for 90 min; CHX, 100 µM, 30 min prior as a pretreatment and during incubation with or without CCCP. (d) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from c. The protein levels in control (nontreated) cells were set as 1. (e) SH-SY5Y cells were pretreated with CHX before the treatments of CCCP, as indicated on top. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 20 µM for 2 h; CHX, 100 µM, 30 min prior as a pretreatment and during incubation with or without CCCP. Molecular mass is indicated in kilodaltons next to the gel blots. (f) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from e. The protein levels in control (nontreated) cells were set as 1.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Expressing, Control, shRNA, Standard Deviation, Generated, Stable Transfection, Transfection, Incubation

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: p97 recruitment to ubiquitinated mitochondria mediates PINK1–Parkin-induced mitochondrial elimination. (a) MEFs from PINK1 +/+ or PINK1 −/− mice were transiently transfected with YFP-Parkin and Myc-p97. Cells were treated with DMSO or CCCP, then immunostained with anti-Myc and anti–cytochrome c . CCCP, 20 µM for 2 h. Bar, 10 µm. (b) HeLa cells transiently expressing Myc-p97 or YFP-Parkin/Myc-p97 were treated with DMSO or CCCP. Cells were immunostained with anti-Myc and anti-Ub (fk1). CCCP, 10 µM for 90 min. The boxed regions are magnified in the bottom panels. (c) HeLa cells were transiently transfected with YFP-Parkin and wild-type Myc-p97 or Myc-p97 QQ . After treatment with CCCP for 24 h, cells were fixed and immunostained with anti-Myc and anti-Tom20 antibodies. CCCP, 10 µM. The boxed regions are magnified in the bottom panels. Bars: (top panels) 10 µm; (bottom enlarged panels) 1 µm. (d) Parkin-induced mitophagy in cells shown as in c was quantified ( n > 50). CCCP, 10 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (e) HeLa cells or HeLa cells expressing YFP-Parkin were treated with CCCP or CCCP plus MG132. Mitochondria were immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 10 µM for 24 h; MG132, 30 µM 30 min prior and with CCCP. Bar, 10 µm. (f) MG132 blocks Parkin-induced mitophagy in HeLa cells as in d. A quantification is shown. Data represent the mean ± SD with P values of at least three replicates.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: Mitochondrial fission is required for Parkin-mediated mitophagy. (a) MEFs from DRP1 +/+ or DRP1 −/− mice were transiently transfected with YFP-Parkin. Cells were treated with CCCP and then immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 20 µM. Bar, 10 µm. (b) Whole cell lysates from DRP1 +/+ or DRP1 −/− MEFs with or without transient expression of Parkin were subjected to SDS-PAGE. Molecular mass is indicated in kilodaltons next to the gel blots. (c) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from b. The protein levels in control (nontreated, nontransfected) cells were set as 1. (d) Clumping mitochondria in DRP1 +/+ or DRP1 −/− MEFs transiently expressing mCherry-Parkin and Mito-YFP were subjected to the FRAP assay. The curve represents an average of 30 individual FRAP curves. CCCP, 20 µM for 8 h. (e) The mobile fractions of Mito-YFP in d. (f and g) YFP-Parkin translocation (f) and YFP-Parkin–induced mitophagy (g) in DRP1 +/+ or DRP1 −/− MEFs treated with CCCP as in panel a were quantified ( n > 50). CCCP, 20 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (h) HeLa cells treated with DRP1 shRNA or control HeLa cells were transiently transfected with YFP-Parkin and treated with or without CCCP. Mitochondria were immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 10 µM for 24 h. Bar, 10 µm. (i) YFP-Parkin–induced mitophagy in HeLa cells with or without DRP1 RNAi as in h was quantified ( n > 50). CCCP, 10 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (j and k) YFP-Parkin–induced mitophagy in MFN1 +/+ 2 +/+ MEFs or MFN1 −/− 2 −/− MEFs was observed (j) and quantified (k) ( n > 50). CCCP, 20 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. Cells expressing YFP-Parkin are indicated with asterisks. (l) A model for Parkin-mediated Mfn degradation and mitophagy. Depolarized mitochondria (orange) that accumulated PINK1 (gray) are sensed by Parkin (green). After Parkin recruitment to the damaged mitochondria, mitochondrial Parkin induces mitofusin (blue) ubiquitination and degradation. Degradation of mitofusin is regulated by p97 (purple) and is important to prevent impaired mitochondria from fusing with the vital mitochondrial network. Damaged mitochondria isolated by Parkin are engaged with autophagosomes and eliminated by auto-lysosomes.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, SDS Page, Standard Deviation, Generated, Control, FRAP Assay, Translocation Assay, shRNA, Ubiquitin Proteomics, Isolation

Journal: Annals of Translational Medicine

Article Title: Propofol protects hippocampal neurons in sleep-deprived rats by inhibiting mitophagy and autophagy

doi: 10.21037/atm-21-3872

Figure Lengend Snippet: Detection of mitophagy-related protein and gene expression. (A-C) Relative expression levels of LC3, Parkin, and PINK1 genes by qRT-PCR. (D-I) Assessment of LC3, PINK1, and Parkin proteins by immunohistochemistry. (J-O) Assessment of autophagy-related and mitophagy-related proteins by Western blotting. *P<0.05, significantly different from the control group; #P<0.05, significantly different from the SD group.

Article Snippet: Sections were then incubated with a rabbit LC3A/B monoclonal antibody (1:500, Cell Signaling Technology, USA), a rabbit Pink1 polyclonal antibody (1:100, Affinity Biosciences. the USA), and a rabbit PARK2/Parkin polyclonal antibody (1:200, Proteintech Group, USA) at 4 °C overnight.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot, Control

Journal: Annals of Translational Medicine

Article Title: Siderophores induce mitophagy-dependent apoptosis in platelets

doi: 10.21037/atm-20-4861

Figure Lengend Snippet: Siderophores induce mitophagy of platelets by activating PINK1/Parkin- and BNIP3-dependent pathways. (A) Washed platelets from healthy volunteers were resuspended in Tyrode’s solution and incubated with 5, 10, 25, or 50 µM enterobactin and 10 µM DFO. In two groups, platelets were incubated with 0.5 µM Baf A1 for 1 hour before incubation with the reagents mentioned above. The expressions in platelets of PINK1 and BNIP3 were analyzed by western blot. GAPDH served as the loading control. (B,C) Quantification analysis of the above proteins in platelets. (D) Washed platelets were incubated with 10 µM enterobactin 10 µM yersiniabactin, 10 µM salmochelin and 10 µM aerobactin. The expressions in platelets of PINK1 and BNIP3 were analyzed by western blot. GAPDH served as the loading control. (E,F) Quantification analysis of the above proteins in platelets. All these images are representative of three independent experiments. All the statistical data were expressed as the means with standard deviations, and statistics were calculated using ANOVA analysis. Statistical significance is showed as follows: *, P<0.05; **, P<0.01; ***, P<0.001. DFO, desferrioxamine.

Article Snippet: The following antibodies were used for western blot: rabbit polyclonal anti-LC3 (Cell Signaling Technology), rabbit polyclonal anti-p62 (ProteinTech), rabbit polyclonal anti-TOMM20 (Cell Signaling Technology), rabbit polyclonal anti-TIMM23 (Cell Signaling Technology), rabbit polyclonal anti- PTEN-induced putative kinase 1 (PINK1) (Cell Signaling Technology) and rabbit polyclonal anti-BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) (Cell Signaling Technology).

Techniques: Incubation, Western Blot