Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 1. Putative functional domains and motifs of PINK1 and conserved subdomains of the PINK1 kinase domain. (A) PINK1 sequence consists of (i) a mitochondrial targeting sequence, (ii) a putative transmembrane domain and (iii) a putative protein serine/threonine kinase domain. The positions of amino acid missense mutations (single-letter code) associated with familial forms of PD are indicated by each arrow. Non-sense mutations are indicated by X, which denotes the introduction of a premature stop codon. Frameshift mutation is indicated by fsX. All except two of the PD-associated mutations are mapped to the protein kinase domain of PINK1. (B) Multiple alignments of the catalytically critical motifs of PINK1 orthologues and other protein kinases. PD-associated A168P mutation resides in the conserved ATP-anchoring b-hairpin P-loop (GXGXXGXV). Two PD-associated mutations were chosen in this study—G386A and G409V mutations. Gly-386 corresponds to the glycine residue in the DFG motif at the start of the activation segment, which is responsible for chelating the Mg2þ ion in the active site. Gly-409 is mapped to the protein substrate-binding loop upstream of the conserved APE motif at the end of the activation segment. ‘Start’ and ‘End’ denote the residues at the N-terminal and C-terminal boundaries of the kinase domain, respectively. (C) Schematic representation of PINK1 constructs generated for expression in Sf9 cells. Introduction of 6-histidine tag and FLAG-tag facilitates purification of the PINK1 proteins by Ni-NTA affinity column chromatography and immunoprecipitation by anti-Flag antibody, respectively.

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Functional Assay, Sequencing, Mutagenesis, Residue, Activation Assay, Binding Assay, Construct, Generated, Expressing, FLAG-tag, Column Chromatography, Immunoprecipitation

Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 2. Western blot and SDS–PAGE analyses of purified recombinant PINK1 proteins used in the enzymatic characterization. (A) PINK1(KD), KD(G386A) and KD(G409V) were purified sequentially by nickel–NTA agarose affinity chromatography and immunoprecipitated prior to enzymatic analysis. PINK1(KD þ T) was purified sequentially by DEAE anion exchange chromatography and immunoprecipitated prior to enzymatic analysis. The purified preparations were analyzed by immunoblotting with anti-PINK1 antibody. The recombinant PINK1 proteins were immunoprecipitated as intact proteins as follows: PINK1(KD þ T), 48.3 kDa; PINK1(KD) and its PD-associated mutants, 42 kDa. (B) The purity of the final preparation of PINK1(KD) was analyzed by silver staining of the SDS–PAGE gel. A single prominent protein band of 42 kDa was detected.

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Western Blot, SDS Page, Recombinant, Chromatography, Immunoprecipitation, Silver Staining

Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 3. Analysis of the substrate preference and amino acid selectivity of PINK1(KD) and its mutants. (A) Comparison of the extent of phosphorylation of histone H1 and casein by PINK1(KD) and its PD-associated mutants. Auto- radiogram of casein phosphorylated by PINK1(KD) and its PD-associated mutants (left). Autoradiogram of histone H1 phosphorylated by PINK1(KD) and its PD-associated mutants (right). On the basis of densitometry compari- son, the level of histone H1 phosphorylation by PINK(KD) is 8-fold higher than that of casein. (B) Phosphoamino acid analysis of histone H1 phosphory- lated by PINK1(KD). The radioactively phosphorylated histone H1 was subject to acid hydrolysis. The hydrolysate was mixed with the phosphoamino acid standards, spotted onto a TLC plate before electrophoresis. Ninhydrin- stained phosphoamino acid standards on the TLC plate (left). Autoradiogram of the TLC plate (right).

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Comparison, Phospho-proteomics, Phosphoamino Acid Analysis, Electrophoresis, Staining

Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 4. PD-associated G386A and G409V mutations reduce PINK1 kinase activity. (A) Mutation of Gly-386 and Gly-409 is expected to cause reduction in the PINK1 kinase activity: Gly-386 and Gly-409 of PINK1 reside in the activation loop, which governs both the activity and substrate recognition of many protein kinases. On the basis of the sequence alignment between PKA and PINK1, Gly-386 and Gly-409 of PINK1 falls within the conserved subdomains VII (blue) and VIII (pale green) of PKA crystal struc- ture (PDB ID: 1ATP). Gly-186 of PKA (equivalent to Gly-386 of PINK1) is hydrogen bonded to Asp-184 of PKA (equivalent to Asp-384 of PINK1) to stabilize its electrostatic interaction with Mg2þ-ATP. Gly-200 of PKA (equivalent to Gly-409 of PINK1) is hydrogen bonded to the backbone of the protein substrate analog, which dictates substrate recognition and binding. These interactions are important for bringing g-phosphate of ATP and the target hydroxyl group in the substrate [represented by the methyl group (magenta) of the substrate analog] together in close proximity for the phosphorylation reaction. Therefore, mutations in Gly-386 and Gly-409 of PINK1 are likely to disrupt the ATP and substrate binding, respectively. Green-dotted lines indicate hydrogen bonds. The image was generated with the Swiss pdb viewer. (B) Purified PINK1(KD) (9.2 pmol), KD(G386A) (20 pmol) and KD(G409V) (35 pmol) were incubated with 15 mg histone H1 in the presence of kinase assay buffer and 5 mM [g-32P]ATP. The extent of histone H1 phosphorylation over time is shown by autoradiograms. (C) The protein bands corresponding to histone H1 were excised and the extent of phosphorylation was quantified and expressed in picomoles of 32P incorporated in histone H1 per micromole of recombinant PINK1 protein and the data were plotted over time. The specific kinase activity of PINK1(KD), KD(G386A) and KD(G409V) was determined from the data presented to be 25, 1.2 and 3.4 pmol of 32P incorporated per minute per micromole of recombinant PINK1 protein, respectively.

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Activity Assay, Mutagenesis, Activation Assay, Sequencing, Binding Assay, Phospho-proteomics, Generated, Incubation, Kinase Assay, Recombinant

Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 5. The effect of C-terminal tail on PINK1 kinase activity. Purified PINK1[KD] (9.2 pmol) and PINK1(KD þ T) (6.9 pmol) were incubated with 15 mg histone H1 in the presence of kinase assay buffer and 5 mM [g-32P]ATP. (A) The extent of histone H1 phosphorylation over time is shown by autoradiograms. (B) The protein bands corresponding to histone H1 were excised and the extent of phosphorylation was quantified and expressed in picomoles of 32P incorporated in histone H1 per micromole of recombinant PINK1 protein and the data were plotted over time. The specific kinase activity of PINK1(KD) and PINK1(KD þ T) were calculated to be 25 and 140 pmoles 32PO4 ¼ incorporated per minute per micromole of recombi- nant PINK1 protein, respectively.

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Activity Assay, Incubation, Kinase Assay, Phospho-proteomics, Recombinant

Journal: Human molecular genetics

Article Title: C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1.

doi: 10.1093/hmg/ddl398

Figure Lengend Snippet: Figure 6. The C-terminal tail influences PINK1’s selectivity of phosphoryl- ation sites in histone H1 but not casein. (A) Tryptic phosphopeptide map of casein phosphorylated by PINK1(KD). (B) Tryptic phosphopeptide map of casein phosphorylated by PINK1(KD þ T). (C) Tryptic phosphopeptide map of histone H1 phosphorylated by PINK1(KD). (D) Tryptic phosphopeptide map of histone H1 phosphorylated by PINK1(KD þ T). (E) A mixture of the samples analyzed in (C) and (D). (F) Schematic of the phosphopeptide maps shown in (C). (G) Schematic of the phosphopeptide maps shown in (D). O: Origin. The arrows in (G) indicate the phosphopeptide fragments that were preferentially phosphorylated by PINK1(KD þ T).

Article Snippet: The human PINK1 cDNA clone was from OriGene Technology.

Techniques: Phospho-proteomics

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 deficiency aggravates kidney dysfunction, albuminuria, and renal morphological changes in the kidneys of diabetic mice. (A) Western blotting of PINK1 in whole kidney lysate obtained from Pink1 +/+ and Pink1 -/- mice with normal or high blood glucose levels. ( B ) % of HbA1c during a 16-week period. (C and D) Levels of serum creatinine ( C ) and blood urea nitrogen ( D ) across groups. ( E ) Quantification of 24-hour albuminuria. ( F ) Representative photomicrographs of periodic acid-Schiff-stained kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. ( G ) A semiquantitative assessment of the tubular injury score was performed. ( H ) Representative photomicrographs of Masson trichrome-stained kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. ( I ) A semiquantitative assessment of renal fibrosis was performed. Scale bar: ( F and H ) non-magnified images (upper panels) = 100 μm, magnified images (lower panels) = 50 μm. Values are expressed as mean ± standard error. n = 7 per group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01, ### p < 0.001 vs Pink1 -/- NG, † p < 0.05, †† p < 0.01 vs Pink1 +/+ DM. Abbreviations : PINK1, PTEN-induced serine/threonine kinase 1; HbA1c, glycated hemoglobin; NG, normoglycemia; DM, diabetes mellitus; PAS, periodic acid-Schiff; MT, Masson trichrome.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Western Blot, Staining

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 deficiency increases the expression of profibrotic molecules in the kidneys of diabetic mice. ( A and B ) mRNA levels ( A ) and western blotting ( B ) of α-SMA, fibronectin, E-cadherin, and TGF-β1 in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. ( C ) Representative immunohistochemical staining of α-SMA and fibronectin in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. ( D and E ) A semiquantitative assessment of the staining intensity in the renal tubules was performed. Quantitative analysis of Figure B are shown in . ( C ) Scale bar = 100 μm. Data are representative of three independent experiments and is expressed as means ± standard error. n = 7 per group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01 vs Pink1 -/- NG, † p < 0.05 vs Pink1 +/+ DM. Abbreviations : PINK1, PTEN-induced serine/threonine kinase 1; α-SMA, alpha-smooth muscle actin; TGF-β1, transforming growth factor-β1; NG, normoglycemic; DM, diabetes mellitus; IHC, immunohistochemistry.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 regulates the profibrotic phenotypes of renal tubular epithelial cells under high glucose conditions. (A) mRNA levels of Pink1 , α-SMA, fibronectin, and TGF-β1 in human renal proximal tubular cells (HKC-8) after treatment with 5- or 30-mM D-glucose. (B) Western blotting of PINK1, α-SMA, fibronectin, and TGF-β1 in HKC-8 cell lysates after treatment with 5- or 30-mM D-glucose. (C) Quantification of the expression of Pink1 , α-SMA, fibronectin, and TGF-β1 mRNAs in HKC-8 cells with control siRNA or Pink1 siRNA under 5- or 30-mM D-glucose. (D) Western blotting of PINK1, α-SMA, and fibronectin in control or PINK1 siRNA-treated HKC-8 cells under 5- or 30-mM D-glucose. (E) Quantification of the expression of Pink1, α-SMA, fibronectin, and TGF-β1 mRNA in primary renal tubular epithelial cells of Pink1 +/+ and Pink1 -/- mice after treatment with 5- or 30-mM D-glucose. (F) Western blotting of α-SMA, fibronectin, and TGF-β1 in 5- or 30-mM D-glucose-treated primary renal tubular epithelial cells obtained from Pink1 +/+ or Pink1 -/- mice. (G) Quantification of the expression of Pink1 , α-SMA, fibronectin, and TGF-β1 mRNA in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. (H) Western blotting of α-SMA, fibronectin, and TGF-β1 in PINK1-overexpressed HKC-8 cells after treatment with 5- or 30-mM D-glucose. Quantitative analysis of Figure B, 3D, 3F, and 3H are shown in . Shown are representatives of three independent experiments and are expressed as means ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01 vs Pink1 -/- NG, †† p < 0.01, ††† p < 0.001 vs Pink1 +/+ DM. Abbreviations: PINK1, PTEN-induced serine/threonine kinase; α-SMA, alpha-smooth muscle actin; TGF-β1, transforming growth factor-β1; NG, normal glucose; DM, diabetes mellitus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; OE, overexpression; GFP, green fluorescent protein.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Western Blot, Expressing, Control, Infection, Over Expression

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 maintain mitochondrial homeostasis and mitophagic function in renal tubular epithelial cells under high glucose conditions. (A) Representative electron microscopy images and the quantification of mitochondrial area and length in renal proximal tubular epithelial cells of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. (B) Western blotting of mitofusin 1 (Mfn1), mitochondrial fission 1 (Fis1), and dynamin-related protein 1 (Drp1) in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels, and their quantification. (C) The mitochondrial membrane potential of HKC-8 cells with Pink1 siRNA and GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. (D) The mouse mitochondrial DNA 3,860-bp deletion in primary cultures of kidney proximal tubule cells obtained from Pink1 +/+ or Pink1 -/- mice and the human mitochondrial DNA 4,977-bp deletion in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. (E) Western blotting of Parkin and Bcl-2/adenovirus E1B 19 kD-interacting protein 3 (BNIP3) in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels, and their quantification. (F) Representative fluorescence images of the mt-Keima and their quantification in HKC-8 cells with control or Pink1 siRNA after treatment with 5- or 30-mM D-glucose. (G) Representative fluorescence images of the mt-Keima and their quantification in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. Scale bar: ( A ) 1 μm and 0.5 μm in upper and lower images, respectively, ( F and G ) 10 μm. ( A, B, and E ) n = 6 per group. (C, D, F, and G) Data are representative of three independent experiments and are expressed as means ± standard error. n = 3 per group. ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01, ### p < 0.001 vs Pink1 -/- , NG, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs Pink1 +/+ DM. Abbreviations: PINK1, PTEN-induced serine/threonine kinase 1; NG, normal glucose; DM, diabetes mellitus; Mfn, mitofusin 1; Fis1, mitochondrial fission 1; Drp1, dynamin-related protein 1; OE, overexpression; mtDNA, mitochondrial DNA; BNIP, Bcl-2/adenovirus E1B 19 kD-interacting protein 3; mt-Keima, mitochondria-targeted Keima.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Electron Microscopy, Western Blot, Membrane, Infection, Control, Fluorescence, Over Expression

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 is involved in mitochondrial electron transport chain complex expressions and cellular oxygen consumption. (A) Western blotting of electron transport chain complex I-V in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. (B-D) Representative Seahorse assay traces of HKC-8 cells with control or Pink1 siRNA after treatment with 5- or 30-mM D-glucose and the quantification of OCR at basal and maximal respiration. (E-G) Representative Seahorse assay traces of control or GFP-Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose and the quantification of OCR at basal and maximal respiration. ( A ) n = 6 per group, (B-G) Data are representative of five independent experiments and are expressed as means ± standard error. ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01, ### p < 0.001 vs Pink1 -/- , NG, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs Pink1 +/+ DM. Abbreviations : PINK1, PTEN-induced serine/threonine kinase 1; NG, normal glucose; DM, diabetes mellitus; OCR, oxygen consumption rate; OE, overexpression.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Western Blot, Control, Infection, Over Expression

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 regulates the generation of reactive oxygen species in renal tubular epithelial cells under high glucose conditions. (A and B) Representative immunohistochemical staining of 8-OHdG in the kidneys of Pink1 +/+ and Pink1 -/- mice with normal or high serum glucose levels. A semiquantitative assessment of the staining intensity in the renal tubules was performed. (C) Representative images of MitoSOX red staining of superoxide radicals and their quantification in HKC-8 cells with control siRNA or Pink1 siRNA after treatment with 5- or 30-mM D-glucose. (D) Immunofluorescence images showing DHE staining and their quantification in HKC-8 cells with control or Pink1 siRNA after treatment with 5- or 30-mM D-glucose. (E) Representative images of MitoSOX red staining of superoxide radicals and their quantification in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. (F) Immunofluorescence images of DHE staining and their quantification in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. Scale bar: ( A ) 100 μm, ( C-F ) 100 μm. ( A ) n = 7 per group, ( C-F ) Data is representative of three independent experiments and expressed as means ± standard error. n = 3 per group. ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, # p < 0.05, ## p < 0.01, ### p < 0.001 vs Pink1 -/- , NG, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs Pink1 +/+ DM. Abbreviations: PINK1, PTEN-induced serine/threonine kinase 1; NG, normal glucose; DM, diabetes mellitus; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; DHE, dihydroethidium; OE, overexpression.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Immunohistochemical staining, Staining, Control, Immunofluorescence, Infection, Over Expression

Journal: International Journal of Biological Sciences

Article Title: PTEN-induced kinase 1 exerts protective effects in diabetic kidney disease by attenuating mitochondrial dysfunction and necroptosis

doi: 10.7150/ijbs.83906

Figure Lengend Snippet: PINK1 inhibits necroptosis in renal tubular epithelial cells under high glucose conditions. (A) Representative immunohistochemical images of TNFα and p-MLKL in the kidneys of Pink1 +/+ and Pink1 -/- with normal or high blood glucose levels. Scale bar = 50 μm. (B) Western blotting of necroptosis-related proteins in primary renal tubular epithelial cells of Pink1 +/+ and Pink1 -/- mice after treatment with 5- or 30-mM D-glucose. (C) Western blotting of necroptosis-related and profibrotic proteins in necrostatin-1-treated HKC-8 cells under 5- or 30-mM D-glucose. (D) Quantification of the mRNA expression of necroptosis-related molecules in HKC-8 cells with control or Pink1 siRNA after treatment with 5- or 30-mM D-glucose. (E) Western blotting of necroptosis-related proteins in control or PINK1 siRNA-treated HKC-8 cells under 5- or 30-mM D-glucose. (F) Quantification of the mRNA expression of necroptosis-related molecules in control or GFP- Pink1 infected HKC-8 cells after treatment with 5- or 30-mM D-glucose. (G) Western blotting of necroptosis-related proteins in PINK1-overexpressed HKC-8 cells after treatment with 5- or 30-mM D-glucose. Quantitative analysis of Figure B, 7C, 7E, and 7G are shown in . ( B-G ) Data are representative of three independent experiments and are expressed as means ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001 vs Pink1 +/+ NG, ### p < 0.001 vs Pink1 -/- NG, †† p < 0.01, ††† p < 0.001 vs Pink1 +/+ DM. Abbreviations: PINK1, PTEN-induced serine/threonine kinase 1; NG, normal glucose; DM, diabetes mellitus; TNFα, tumor necrosis factor α; MLKL, mixed lineage kinase domain-like pseudokinase; RIPK, receptor-interacting protein kinase; α-SMA, alpha-smooth muscle actin; OE, overexpression.

Article Snippet: We used lentiviral expression vector cloned PINK1 human tagged ORF (CAT#: RC206970L4, Origene, Rockville, MD), the pLenti-C-mGFP-P2A lentiviral vector.

Techniques: Immunohistochemical staining, Western Blot, Expressing, Control, Infection, Over Expression

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: Primers for Real-time quantitative PCR.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: Virus infection down regulates PINK1 expression in macrophages. (A) Q-PCR analysis of PINK1 mRNA expression in mouse peritoneal macrophages (PMs) (3 × 10 5 ) infected with VSV (MOI = 1), RSV (MOI = 10), or HSV (MOI = 10), respectively, for indicated hours. (B) Immunoblot analysis of PINK1 protein expression in mouse peritoneal macrophages (3 × 10 6 ) infected with VSV for indicated hours. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). (C) Q-PCR analysis of PINK1 mRNA expression in RAW264.7 cells (1 × 10 5 ) infected with VSV for indicated hours. (D) Q-PCR analysis of PINK1 mRNA expression in peripheral blood mononuclear cells (PBMCs) of 38 pediatric patients with RSV infection and 21 healthy children. Data are mean ± SD. Similar results were obtained in three independent experiments.* p < 0.05, ** p < 0.01, compared with control.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Virus, Infection, Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 knockdown or knockout suppresses virus-induced type I interferon and proinflammatory cytokine production. Mouse peritoneal macrophages (PMs) were transfected with 30 nM scrambled negative control siRNA (siNC) or PINK1 siRNA (siPINK1) for 48 h. PINK1 knockout RAW264.7 macrophages (PINK1 −/− ) were generated using CRISPR/Cas9 gene-editing system. (A) Immunoblot analysis of PINK1 expression level in PMs with PINK1 knockdown, or RAW264.7 cells with PINK1 knockout. (B) qPCR analysis of IFN-β mRNA expression in PMs infected for indicated MOIs with VSV for 6 h. (C–E) qPCR and ELISA analysis of IFN-β and IL-6 levels of in PMs infected with VSV, RSV, or HSV, respectively, for indicated hours. (F) qPCR analysis of IFN-β and IL-6 levels in wild type (PINK1 +/+ ) and PINK1 knockout cells (PINK1 −/− ) RAW264.7 cells infected with VSV for indicated hours. Data are mean ± SD and are representative of three independent results. * p < 0.05, ** p < 0.01, compared with control.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Knockdown, Knock-Out, Virus, Transfection, Negative Control, Generated, CRISPR, Western Blot, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 promotes RLR-triggered IRF3 and NF-κB activation in macrophages. Mouse peritoneal macrophages transfected with 30 nM scrambled negative control siRNA (siNC) or PINK1 siRNA (A) , or RAW264.7 cells transfected with plasmids encoding Myc-PINK1 (B) , were infected with VSV for indicated hours. Phosphorylated or total proteins in lysates were detected by western blot. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Activation Assay, Transfection, Negative Control, Infection, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 interacts with TRAF3 and IRF3 upon VSV infection. (A) Mouse peritoneal macrophages were infected with VSV for indicated hours. Immunoblot analysis of endogenous TRAF3, IRF3, Parkin, RIG-I, TBK1, and MAVS immunoprecipitated with antibody to PINK1. IgG was as control. Numbers between two blots indicate densitometry of TRAF3, IRF3, or Parkin relative to that of PINK1 in immunoprecipitates. (B,C) HEK293T cells were transfected with PINK1 expressing plasmid together with Flag-TRAF3 or HA-IRF3 plasmid. Cells were lysated 24 h after transfection for immunoblot analysis of indicated proteins immunoprecipitated with antibody to Flag (B) or HA (C) tag. (D) Confocal microscopy of HEK293T co-transfected with Myc-PINK1 and Flag-TRAF3 plasmids followed by VSV infection for 4 h. MitoTracker (Mito) was used to probe the mitochondrion (red). DAPI served as a marker of nuclei (blue). Scale bar, 5 μm. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Control, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Marker

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 promotes RLR-triggered immune response via kinase domain dependent manner. (A) Immunoblot analysis of HEK293T cells that cotransfected with GFP-TRAF3 plus Flag-PINK1, Flag-PINK1 mutants vectors followed by VSV infection for 4 h, then immunoprecipitated with antibody to Flag tag. (B) Immunoblot analysis of RAW264.7 cells stably overexpressed with empty vector or vector encoding PINK1 and its mutants. (C) qPCR analysis of IFN-β, IL-6, and VSV-G mRNA in stably transfected RAW264.7 cells expression of PINK1 variants, infected with VSV for indicated times. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Western Blot, Infection, Immunoprecipitation, FLAG-tag, Stable Transfection, Plasmid Preparation, Transfection, Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 inhibits TRAF3 degradation. Mouse peritoneal macrophages transfected with scrambled negative control siRNA (siNC) or PINK1 specific siRNA (30 nM) (A) , or RAW264.7 cells transfected with PINK1 plasmid (B) , were infected with VSV for indicated hours. TRAF3 proteins in lysates were detected by western blot. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). (C) HEK293T cells were transfected with GFP-TRAF3, HA-Ub, Flag-Parkin, and varying doses of Flag-PINK1 (0, 0.5, and 1 μg) and infected with VSV for 4 h. Cells were treated with MG132 (10 uM) and harvested for immunoblot analysis of K48-Ub and K63-Ub immunoprecipitated with antibody to GFP tag. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Transfection, Negative Control, Plasmid Preparation, Infection, Western Blot, Expressing, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 associates with YAP1 and inhibits the interaction of YAP1 with IRF3. (A) Mouse peritoneal macrophages were infected with VSV for indicated hours. Immunoblot analysis of endogenous YAP1 immunoprecipitated with antibody to PINK1. IgG was as control. (B) Confocal microscopy of HEK293T cells that co-transfected with Myc-PINK1 and Flag-YAP1 plasmids followed by VSV infection for 4 h. Scale bar, 5 μm. (C) HEK293T cells were transfected for 24 h with plasmids encoding HA-IRF3, Flag-YAP1, and Myc-PINK1. Immunoblot analysis of indicated proteins immunoprecipitated with antibody to Flag tag. (D) Mouse peritoneal macrophages transfected with scrambled negative control siRNA (siNC) or PINK1 specific siRNA (30 nM) were infected with VSV for indicated hours. Phosphorylated or total proteins of YAP1 in lysates were detected by western blot. Similar results were obtained in three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Control, Confocal Microscopy, Transfection, FLAG-tag, Negative Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 positively regulates the RLR-triggered antiviral immune response by inhibiting TRAF3 degradation and relieving YAP-mediated inhibition of the cellular antiviral response. (1) Virus infection down-regulates PINK1 expression in macrophages. (2) PINK1 promotes RLR-triggered IRF3 activation via inhibiting Parkin-mediated K48-linked ubiquitination of TRAF3, (3) and PINK1 inhibits YAP1/IRF3 complex formation, which ultimately promotes RIG-I triggered antiviral immune response.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Inhibition, Virus, Infection, Expressing, Activation Assay, Ubiquitin Proteomics

Journal: Cell Death Discovery

Article Title: Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson’s disease

doi: 10.1038/cddiscovery.2015.15

Figure Lengend Snippet: Binding of IPAS to PINK1. ( a and b ) Binding of IPAS to PINK1. HeLa cells ( a ) and SH-SY5Y cells ( b ) were transfected with pMyc-IPAS and pPINK1-FLAG, and membrane fractions were isolated. Immunoprecipitation was performed with antibody against FLAG followed by immunoblotting with antibody against Myc. ( c ) Binding of IPAS to endogenous PINK1. SH-SY5Y cells were transfected with pMyc-IPAS and treated with CCCP and MG132 for indicated time periods. After solubilization of membrane fractions, immunoprecipitation was performed using anti-Myc antibody followed by immunoblotting with antibody against PINK1. ns, nonspecific band.

Article Snippet: PINK1-pCMV6 vector (MR209092) was purchased from Origene (Rockville, MD, USA), and used after removal of the DDK-tag sequence to produce pCMV6-PINK1-Myc. pCMV6-PINK1-FLAG was constructed by replacing the Myc-DDK sequence of PINK1-pCMV6 with a FLAG tag.

Techniques: Binding Assay, Transfection, Isolation, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson’s disease

doi: 10.1038/cddiscovery.2015.15

Figure Lengend Snippet: Phosphorylation of IPAS by PINK1. ( a ) Phos-tag-based immunoblot analysis of CCCP-activated IPAS. PINK1 siRNA treated (24 h) SH-SY5Y cells were transfected with pMyc-IPAS for 24 h and treated with CCCP and MG132 as indicated, and membrane fractions were analyzed by Phos-tag immunoblotting using anti-Myc antibody. Band 0 indicates unphosphorylated IPAS. Bands 1, 2, 3 and 4 indicate phosphorylated IPAS. ( b ) Alignment of amino acid sequences containing the putative phosphorylation sites of IPAS and phosphorylation sites of several PINK1 substrates. The putative phosphorylated Thr residue and known phosphorylated Ser or Thr residues are indicated by an arrow and arrowhead, respectively. Hydrophobic amino acids found two residues after phosphorylation sites are boxed, and basic amino acids are designated in bold. ( c ) Phosphorylation of Thr12 by PINK1. SH-SY5Y cells were transfected with pMyc-IPAS WT, T12A or S10A, and analyzed by Phos-tag immunoblotting as in a . ( d ) Lack of polyubiquitination of IPAS T12A mutant by Parkin. SH-SY5Y cells were transfected for 24 h with plasmids for IPAS WT, IPAS N, IPAS C or IPAS T12A together with HA-Ub, treated with CCCP and MG132 for 30 min and analyzed by immunoblotting with antibody against HA. ( e ) Lack of binding of IPAS T12A mutant to Parkin. SH-SY5Y cells were transfected with plasmids of IPAS WT or the T12A mutant, and treated with CCCP and MG132 for 2 h. Immunoprecipitation was performed with antibody against FLAG followed by immunoblotting with antibody against Myc. ( f ) Attenuation of IPAS binding to Parkin by PINK1 siRNA treatment. PINK1 siRNA treated SH-SY5Y cells were transfected with pMyc-IPAS and FLAG-Parkin and treated with CCCP and MG132 for 60 min. Immunoprecipitation was performed as in e.

Article Snippet: PINK1-pCMV6 vector (MR209092) was purchased from Origene (Rockville, MD, USA), and used after removal of the DDK-tag sequence to produce pCMV6-PINK1-Myc. pCMV6-PINK1-FLAG was constructed by replacing the Myc-DDK sequence of PINK1-pCMV6 with a FLAG tag.

Techniques: Western Blot, Transfection, Mutagenesis, Binding Assay, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson’s disease

doi: 10.1038/cddiscovery.2015.15

Figure Lengend Snippet: Attenuation of IPAS-induced apoptosis by the activation of the PINK1–Parkin pathway. ( a ) Attenuation of IPAS-induced apoptosis by CCCP treatment. Parkin siRNA treated (24 h) SH-SY5Y cells were transfected with pCerulean-IPAS. Four hours after transfection, cells were treated with CCCP (20 μ M) for 100 min, and cultured for 24 h. Cells were stained with active caspase-3 antibody and Hoechst 33342, and were observed using a fluorescence microscope. A minimum of 300 transfected cells per sample was counted and ratio of active caspase-3 positive cells to Cerulean-IPAS-expressing cells is calculated (right). Data shown in bar graphs are mean±S.D. of three independent experiments. ** P <0.01 for indicated comparison. ( b ) SH-SY5Y cells were transfected with pCerulean, pCerulean-IPAS, pCerulean-IPAS+pMyc-mParkin WT, pCerulean-IPAS+pMyc mParkin T415N or pCerulean-IPAS+pMyc-hParkin, stained as in a , and observed using a fluorescence microscope. Representative images of transfected cells are shown in . A minimum of 100 transfected cells per sample were processed as in a . Data shown in bar graphs are mean±S.D. of three independent experiments. * P <0.05.

Article Snippet: PINK1-pCMV6 vector (MR209092) was purchased from Origene (Rockville, MD, USA), and used after removal of the DDK-tag sequence to produce pCMV6-PINK1-Myc. pCMV6-PINK1-FLAG was constructed by replacing the Myc-DDK sequence of PINK1-pCMV6 with a FLAG tag.

Techniques: Activation Assay, Transfection, Cell Culture, Staining, Fluorescence, Microscopy, Expressing