Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Identification of the heat shock proteins HSP70 and HSP90 as putative ChAT protein-interactors by proximity-dependent biotin identification (BioID). (A) Optimization of BioID in HEK293 cells expressing wild-type or P17A/P19A-ChAT fused to the HA-tagged promiscuous biotin ligase BirA-R118G (BirA * ). Control cells were transfected with empty vector or plasmids encoding either untagged ChAT or BirA * . Cells were treated for 24 h with either 50 μM biotin to facilitate proximity-dependent biotinylation of ChAT-interacting cellular proteins or with vehicle-control (water). Biotinylated proteins were isolated from cell lysates by streptavidin pull-downs (PD: Strep) and immunoblotted as indicated ( n = 2). (B) Identification of HSP70 and HSP90 as ChAT proximally-interacting proteins. Streptavidin PD samples prepared from biotin-treated HEK293 cells expressing wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * fusion proteins were resolved and visualized on a silver-stained SDS-PAGE gels. Two proteins (~70 and ~90 kDa) that were enriched in samples expressing P17A/P19A-ChAT-BirA * were identified by MALDI-TOF-MS or LC-ESI-MS/MS as HSP70 and HSP90, respectively. Control cells were transfected to express untagged wild-type ChAT ( n = 1). (C) Confirmation of endogenous HSP70 and HSP90 as putative ChAT-interacting proteins by immunoblotting of streptavidin PD samples prepared from biotin-treated HEK293 cells expressing HA-tagged wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * . Control cells were transfected with empty vector or vector encoding untagged ChAT ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Staining, SDS Page, Tandem Mass Spectroscopy, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSP90 ATPase activity specifically reduces steady-state levels of mutant ChAT protein. Immunoblots from SN56 cells expressing wild-type (A) , P17A/P19A- (B) , V18M- (C) , or A513T-ChAT (D) that were treated for 24 h with 0.5–2 μM with 17-AAG, an inhibitor of HSP90 activity, or with DMSO-vehicle. (E) Treatment of cells with 17-AAG at concentrations up to 2 μM has no effect on the steady-state levels of wild-type ChAT protein. Conversely, steady-state protein levels of P17A/P19A- (F) , V18M- (G) , and A513T-ChAT (H) are reduced following treatment of cells with 17-AAG compared to vehicle-control ( *** p ≤ 0.001; one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 4). (I) Proteasome inhibition by co-treatment with 5 μM MG132 for 18 h attenuates the effects of inhibition of HSP90 (1 μM 17-AAG, 24 h) on ChAT steady-state protein levels in ChAT-expressing SN56 cells. Control cells were treated with DMSO-vehicle. Proteasome inhibition was validated by immunoblotting for the accumulation of ubiquitinated cellular proteins ( n = 4). (J) Lysosomal inhibition by co-treatment with 50 μM chloroquine (CQ) for 18 h did not prevent the effects of 17-AAG treatment (1 μM, 24 h) on steady-state ChAT protein levels in SN56 cells. Lysosomal inhibition was validated by immunoblotting for the accumulation of the lysosome-associated protein LC3B-II ( n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Inhibition, Activity Assay, Mutagenesis, Western Blot, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSC/HSP70 and HSP90 activity enhances ChAT ubiquitination and reduces cellular ChAT enzymatic activity. (A) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for 24 h with 40 μM VER-155008 and for the final 6 h with 20 μM MG132 to inhibit degradation of ubiquitinated ChAT. Control cells were transfected with empty vector and/or treated with DMSO-vehicle. Inhibition of HSC/HSP70 by VER-155008 treatment enhanced ChAT ubiquitination, where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (B) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for either 8 or 24 h with 1 μM 17-AAG and for the final 6 h with 20 μM MG132. Inhibition of HSP90 by treatment with 1 μM 17-AAG for 8 h, but not for 24 h, enhanced ChAT ubiquitination where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (C) Cellular activity of wild-type ChAT is reduced following treatment of SN56 cells for 24 h with either 40 μM VER-155008 or 1 μM 17-AAG compared to vehicle-treated (DMSO) cells ( ** p ≤ 0.01). Treatment with VER-155008 trended toward a greater reduction in ChAT activity compared to 17-AAG ( p = 0.074; one-way ANOVA with Tukey's post-hoc test, mean ± SEM, n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Oligonucleotide sequence for primers used in ChIP-qPCR analysis for TFEB binding to SMPD1 promoter.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: SMPD1 is a direct target gene for TFEB. (A) Western blots and densitometric analysis of TFEB, LAMP-1 and SMPD1 in JEG3 cells following transient transfection with TFEB siRNA or a control (ss) scrambled sequence ( N = 3 separate experiments run in duplicate; * P < 0.05 compared to ss control). (B) Luciferase reporter assay showing SMPD1 expression in JEG3 cells following overexpression of TFEB (OE TFEB) or empty vector (OE EV). ( N = 3 separate experiments; * P < 0.05 compared to empty vector control). (C) qPCR of SMPD1 promoter regions –200 to 300 bp and –900 to 1,000 bp after chromatin immunoprecipitation with TFEB in E-PE and PTC placentae ( N = 4 for each group). (D) qPCR of SMPD1 promoter region –200 to 300 bp after chromatin immunoprecipitation with TFEB in JEG3 cells exposed to 20 μM CER16:0 or EtOH vehicle.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Western Blot, Transfection, Control, Sequencing, Luciferase, Reporter Assay, Expressing, Over Expression, Plasmid Preparation, Chromatin Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Ceramide triggers lysosomal exocytosis in JEG3 cells. (A) IF images for LAMP-1 and SMPD1 in JEG3 cells following exposure to 20 μM CER 16:0 or EtOH vehicle. (B) IF images of FITC-dextran (green) loaded JEG3 cells following exposure to 20 μM CER 16:0 or EtOH vehicle. Nuclei: DAPI (blue). (C) Fold change in fluorescence intensity of FITC-dextran in JEG3 cells treated with 20 μM CER 16:0 or EtOH vehicle ( N = 3 separate experiments; * P < 0.05 compared to vehicle). (D) Fold change in fluorescence intensity of released FITC-dextran in media of JEG3 cells treated with 20 μM CER 16:0 or EtOH vehicle ( N = 3 separate experiments; ** P < 0.01 compared to vehicle). (E) Fold change variation of intracellular Ca 2+ content of JEG3 cells treated with 20 μM CER 16:0 versus EtOH vehicle. Data are expressed as mean ± SEM.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: L-SMPD1 localizes to lipid rafts of apical syncytial membranes in E-PE placentae and is released into the maternal circulation via exosomes. (A) IF images depicting LAMP-1 and ceramide localization in E-PE and PTC placentae. LAMP-1 (green); Ceramide (red); nuclear DAPI (blue). ST, syncytiotrophoblast. (B) WB and associated densitometry for L-SMPD1 in lysates of apical syncytial membranes (AM) of PTC and E-PE placentae (PTC, N = 3; PE, N = 5; ** P < 0.01 vs. PTC). PLAP was used as AM marker. (C) Total ceramide levels, measured by LC-MS/MS, in syncytial membranes extracts of TC and E-PE placentae ( N = 3 placentae per group; * P < 0.05 compared to control). ( D-left panel ) Distribution of L-SMPD1 in detergent insoluble (Ins) and soluble (Sol) fractions of AM from TC and E-PE placentae. PLAP was used as lipid raft (detergent insoluble fraction) marker. ( D-right panel ) Total ceramide levels measured by LC-MS/MS in extracts of AM Ins and Sol fractions of TC and E-PE placentae ( N = 3 samples per group; * P < 0.05 compared to TC). (E) WB for L-SMPD1, CD63 and PLAP of exosomes isolated from PTC ( N = 3) and E-PE ( N = 3) maternal plasma. (F) WB for L-SMPD1 in PLAP-precipitated exosomes from PTC ( N = 3) and E-PE ( N = 3) maternal plasma. ( F-right panel ) LC-MS/MS quantification of total ceramide in PTC and E-PE PLAP-precipitated exosomes (E-PE, N = 4; PTC, N = 3; * P < 0.05 vs. to PTC). Data are expressed as mean ± SEM. Dotted line: non-contiguous lanes run on the same gel.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Marker, Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Clinical Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: SMPD1 enriched exosomes induces endothelial cells activation. (A) WB for L-SMPD1, CD63 and corresponding densitometry of exosomes isolated from conditioned media of JEG3 cells treated with 20 mM CER 16:0 or EtOH ( N = 3 separate experiments). (B) SMPD1 enzyme activity of exosomes from conditioned media of JEG3 cells treated with CER 16:0 or EtOH ( N = 4 separate experiments). RFU, relative fluorescence units. (C) Representative WB for L-SMPD1 of exosomes from JEG3 cells treated with CER 16:0 or EtOH vehicle in conjunction with TFEB siRNA or control (ss) scrambled sequence treatment ( N = 3 separate experiments). (D) WB and densitometry of ICAM-1 in lysates of HMVEC cells treated for 3 h with ExoCer or ExoV ( N = 3 separate experiments). (E) IF images and mean fluorescence intensity quantification of ICAM-1 (Green) in HMVEC cells treated with ExoCer or ExoV ( N = 3 separate experiments). Nuclei: DAPI (blue). ( F-left panels) Representative flow cytometry density plots of CD54 (ICAM-1) and CD146 of HMVEC cells exposed for 3 h to either 2 × 10 6 ExoV or ExoCer, or 20 mM CER 16:0. ( F-right panel ) Quantification of CD54 + /CD146 + HMVEC cells after treatments ( N = 4 experiments). (G) Densitometry of WB for ICAM-1 normalized to stain free gel in HMVEC lysates treated for 3 h with ExoV or ExoCer in presence or absence of 25 μM Imipramine (I) ( N = 3 separate experiments).

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Activation Assay, Isolation, Activity Assay, Fluorescence, Control, Sequencing, Flow Cytometry, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Active SMPD1 in exosomes from JEG3 cells exposed to ceramide affect endothelial angiogenesis. (A) Angiogenesis assay of HMVEC treated with 2 × 10 6 ExoV or ExoCer ( N = 4 separate experiments). Tube formation was documented after 3 h of treatment by quantification of number of branches and the total length of the segments of the network. Arrows indicates the main branches. Data are expressed as mean ± SEM ( N = 4 separate experiments). (B) Tube formation assay of HMVEC cells treated for 3 h with ExoV or ExoCer in presence or absence of 25 μM Imipramine (I) or 10 μM Fluoxetine (F) and accompanying quantification (C) of tubular branches. Data are expressed as mean ± SEM ( N ≥ 3 separate experiments).

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Angiogenesis Assay, Tube Formation Assay

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) Human FGF-21 is cleaved by FAP. Recombinant human FGF-21 was digested by recombinant human FAP and visualized by Coomassie staining of SDS-Page gel. (B) Time course of FGF-21 digestion by FAP quantified by LC/MS extracted ion integration of peaks corresponding to intact (1–181) and cleaved (1–171) forms of FGF-21 (n = 3 per time point per group). Values are mean ± SEM with one phase decay curve fit superimposed. (C) FAP cleavage of FGF-21 is prevented by ARI-3099. ARI-3099 was pre-incubated with recombinant FAP for 30 minutes prior to addition of FGF-21. Reaction products were visualized by Coomassie staining of SDS-Page gel. (D) Recombinant PREP does not cleave FGF-21. Recombinant human PREP was added to recombinant FGF-21 and visualized by Coomassie staining of SDS-Page gel.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Recombinant, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) FAP cleaves human FGF-21 in mouse, monkey and human plasma. Recombinant FGF-21 was added to plasma to a final concentration of 1 μM in the presence or absence of 16 μM ARI-3099 followed by assessment of intact FGF-21 by sandwich ELISA (n = 3 per group). Values are mean ± SEM. *P < .05 ***P < .001 by ANOVA . (B) FAP activity of mouse, monkey and human plasma as assessed by the FAP-specific fluorescent substrate ARI-3144.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Clinical Proteomics, Recombinant, Concentration Assay, Sandwich ELISA, Activity Assay