pertkr amc fluorogenic peptide substrate  (R&D Systems)

Journal: Communications biology

Article Title: Minimal mechanistic component of HbYX-dependent proteasome activation that reverses impairment by neurodegenerative-associated oligomers.

doi: 10.1038/s42003-023-05082-9

Figure Lengend Snippet: Fig. 1 Identifying residues in the α intersubunit pocket critical for HbYX-dependent gate-opening. a Surface representation of 20S proteasomes in complex with activators [Human 26S (PDB 6msk), yeast 20S+Blm10 (PDB 4v7o), archaeal 20S + PAN (PDB 6hed)]. HbYX motifs visible are colored red- orange and adjacent α-subunits of the visible HbYX motif are shown in various colors. b Surface representation of the 20S α rings (from a) down the center axis with activator caps removed. Proteasome activator C-termini HbYX residues are shown in red-orange (surface). c Overlay of H20S and Y20S intersubunit pockets α5/6 and T20S intersubunit pockets α/α (cartoon) from B with HbYX motif residues (sticks). Crystal structure of PAN C-terminus (PDB 3ipm) is shown in place of Cryo-EM PDB 6hed. Images were rendered with PyMOL. d Multiple sequences alignment of the T20S α subunit with various eukaryotic α6 subunits generated with CLUSTAL OMEGA (1.2.4). e Conserved residues interacting with bound HbYX motif (sticks) in the T20S intersubunit pocket (PDB 3ipm). PAN HbYX motif (LYR) shown in cyan (stick). f Rate of substrate degradation (fluorogenic nonapeptide LFP) by the wild type (WT) T20S proteasome (0.14 nM) or K66/K33/L81 mutants incubated with or without PAN (with ATPγS). Stimulation of gate opening was measured by the increase of LFP hydrolysis (rfu/min) relative to WT 20S without PAN. g Experiments with T20S proteasome (0.35 nM of wild-type or L81Y mutant) performed same as in (f). h Same as (E), with L81 mutated to tyrosine (magenta stick). Images in e and h were rendered with PyMOL. Data (means) are representative of three or more independent experiments each performed in triplicate. Error bars represent ± standard deviation.

Article Snippet: Fluorogenic substrate peptides were purchased from BostonBiochem (suc-LLVY-amc) and EZBiolabs (acnLPnLD-amc, ac-RLR-amc, LFP (Mca-AKVYPYPME-Dpa(Dnp)-amide)), PAN CT peptides, ZYA, and ZYA derivatives were synthesized by ABclonal.

Techniques: Cryo-EM Sample Prep, Generated, Incubation, Mutagenesis, Standard Deviation

Journal: Communications biology

Article Title: Minimal mechanistic component of HbYX-dependent proteasome activation that reverses impairment by neurodegenerative-associated oligomers.

doi: 10.1038/s42003-023-05082-9

Figure Lengend Snippet: Fig. 3 ZYA robustly stimulates gate-opening in yeast and mammalian proteasomes. a Dose response of ZYA with mammalian 20S proteasomes (0.5 nM) and nLPnLD-AMC as substrate. Proteasome activity is normalized to DMSO control. Means were fit to the Hill equation, and equilibrium binding coefficients are shown. b WT and gateless (α3ΔN) yeast 20S proteasomes (0.5 nM) incubated with or without ZYA (2.5 mM) and nLPnLD-amc. c Mammalian 20S proteasomes (0.5 nM) alone or with ZYA (2.5 mM), or PA26 (55 nM), or both (see graph key). Proteasome activity measured using three different fluorogenic substrates preferentially cleaved by different 20S protease sites (LLVY-amc, chymotrypsin-like, nLPnLD- amc, caspase-like; LRR-amc, trypsin-like). Data (means) are representative of three or more independent experiments each performed in triplicate. Error bars represent ± standard deviation.

Article Snippet: Fluorogenic substrate peptides were purchased from BostonBiochem (suc-LLVY-amc) and EZBiolabs (acnLPnLD-amc, ac-RLR-amc, LFP (Mca-AKVYPYPME-Dpa(Dnp)-amide)), PAN CT peptides, ZYA, and ZYA derivatives were synthesized by ABclonal.

Techniques: Activity Assay, Control, Binding Assay, Incubation, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: DUSP2 regulates VEGF-C expression mainly via post-translational modification . (a) Representative Western blots show expression of VEGF-C in conditioned media of control and DUSP2-KD PANC-1 cells at different time points. Angiogenin (ANG) was used as a loading control in conditioned medium. (b) AsPC1-VEGF-C cells were treated with cycloheximide (CHX) and BFA to block protein synthesis and protein secretion. The size of prepro-VEGF-C, pro-VEGF-C and processed VEGF-C was detected as indicated. (c) AsPC1-VEGF-C cells were transiently transfected with GFP or DUSP2-GFP plasmids and levels of DUSP2, VEGF-C in whole cell lysate (WCL) and conditioned medium (CM) were detected. (d) Proprotein convertase activity in control and DUSP2-KD PANC-1 cells. Cell lysates of control and DUSP2-KD cells were incubated with two different concentrations of fluorogenic proprotein convertase substrate. Fluorescent was measured in a kinetic manner with Ex: 360–380 nm, Em: 440–460 nm. Representative quantification (of three independent experiments) is shown. (e) Proprotein convertase activity was measured in control and DUSP2-KD PANC-1 cells treated with proprotein convertase inhibitor for 24 h. (f) Representative images (left) and the quantitative result ( n = 3, right) show loss-of-DUSP2-enhanced VEGF-C secretion was inhibited by treatment with proprotein convertase inhibitor. Control and DUSP2-KD PANC-1 cells were treated with proprotein convertase inhibitor (20 μM) in serum-free RPMI medium. After 24 h, serum-free conditioned media were collected and VEGF-C expression was measured. ** P < 0.01 compared to control, # P < 0.05 compared to DUSP2-KD. (g) Control and DUSP2-KD cells were pre-treated with proprotein convertase inhibitor and plated for invasion ability. Recombinant VEGF-C was treated in the upper chamber of transwell. * P < 0.05.

Article Snippet: Protein convertase substrate (R&D pERTKR-AMC # ES013) was pre-warmed (15 min at 37°C) and then incubated with cell lysate in black 96 well plates.

Techniques: Expressing, Modification, Western Blot, Control, Blocking Assay, Transfection, Activity Assay, Incubation, Recombinant

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: DUSP2 regulates the secretion of EV-VEGF-C in pancreatic cancer cells . (a) Representative (upper) and quantification (bottom) of VEGF-C and HSP70 expression by Western blotting in EV (isolated by ExoQuick-TC) from control and DUSP2-KD PANC-1 cells. (b) Representative Western blots show VEGF-C expression in WCL, EV and CM in DUSP2-KD PANC-1 cells (left). Representative Western blots show VEGF-C expression in EV isolated from DUSP2-KD PANC-1 cells treated with DMSO (control) and SCH 772984 (1uM) for 24 h (right). CD63 and HSP70 were detected as EV markers. ALB was detected to demonstrate the purity of EV. Equal amount of protein (20 ug) was loaded. EV was isolated by ExoQuick-TC. (c) Images of control and DUSP2-KD PANC-1 cells transfected with VEGF-C-SNAP tag for 24 h. Cells were treated with proprotein convertase inhibitor in serum-free medium for another 24 h after transfection. After labelling by the fluorescent substrate for SNAP, cells were fixed and imaged. (d) Images of control and DUSP2-KD PANC-1 cells taken by the transmission electron microscope. Red arrows indicate positive staining by 10 nm gold labelled anti-VEGF-C antibody. (e) Nanoparticle Tracking Analysis (NTA) was performed to detect secreted particles from control and DUSP2-KD cells. Serum-free conditioned medium was collected and centrifuged to remove debris and were sent for NTA. The X-axis represents the size of particles and the Y-axis represents the number of particles secreted per cell. DUSP2-KD cells have increased numbers of EV ranging from 85 to 155 nm (middle panel). EV secreted from DUSP2-KD cells was diminished if treated with GW4869 (20 μM) (right panel). (f) Inverted light sheet microscope (Luxengo) was used to track fast-moving particles in control and DUSP2-KD cells. Cells were labelled with PKH67 and plated into the cell holder for the tracking of PKH67 positive particles within the cells. 3D tracking of PKH67 particles in control and DUSP2-KD cells analysed by Imaris software (left). Particles in DUSP2-KD cells have increased speed (upper right) and track length (bottom right). Experiments have been performed two times and represented data are shown. (g) DUSP2-KD cells have an increased rate of EV-VEGF-C secretion. VE-Snaptag was stably expressed in control and DUSP2-KD cells. Cells were treated with proprotein convertase inhibitor (20 uM) in serum-free RPMI. EV was isolated from the medium at a different time point. Western blotting was performed to detect unprocessed VEGF-C (VE-Snap) in EV (upper). Expression of EV-VEGF-C in control and DUSP2-KD cells by fold change analysis over 2 h (lower). (h) Schematic of experimental design to investigate the function of EV from DUSP2-KD PANC-1 cells (left). Representative immunohistochemistry images show the increase of lymphatic vessels (Lyve-1) in PANC-1 tumours treated with EV isolated from DUSP2-KD cells.

Article Snippet: Protein convertase substrate (R&D pERTKR-AMC # ES013) was pre-warmed (15 min at 37°C) and then incubated with cell lysate in black 96 well plates.

Techniques: Expressing, Western Blot, Isolation, Control, Transfection, Transmission Assay, Microscopy, Staining, Software, Stable Transfection, Immunohistochemistry

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: A proposed model shows the role of DUSP2-VEGF-C axis in promoting pancreatic cancer progression . Downregulation of DUSP2 in pancreatic cancer cells leads to ERK phosphorylation which enhances proprotein convertase activity. Therefore, the production of a functional form of VEGF-C is increased. On the other hand, the amount and moving ability of EV are increased in DUSP2 knockdown cells, which enhance the secretion of functional VEGF-C to the tumour microenvironment. Increased EV-VEGF-C can promote lymphangiogenesis and enhances pancreatic cancer cell invasive ability, leading to lymphovascular invasion.

Article Snippet: Protein convertase substrate (R&D pERTKR-AMC # ES013) was pre-warmed (15 min at 37°C) and then incubated with cell lysate in black 96 well plates.

Techniques: Phospho-proteomics, Activity Assay, Functional Assay, Knockdown