full length wildtype human lamp1  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Arg177 from the dog PrP strongly slows down liquid–liquid phase separation of full-length human PrP C , but His177 from the human PrP greatly enhances liquid–liquid phase separation of full-length dog PrP C . Samples (50 μM) of bacterial-purified full-length wildtype human PrP C (HoPrP-WT) ( A − C ) and its single variant H177R ( D − F ) as well as bacterial-purified full-length wildtype dog PrP C (CaPrP-WT) ( G − I ) and its single variant R177H ( J − L ) were labeled by TAMRA ( red fluorescence ) ( A , D , G , and J ) and incubated with 1× PBS (pH 7.4) containing 10% (w/v) PEG 8000 on ice to induce LLPS for 10 min. Liquid droplets of the human PrP C or the dog PrP C were observed by differential interference contrast (DIC) confocal microscopy, with excitation at 561 nm. DIC microscopic images, B , E , H , and K . Merge, C , F , I , and L . We have replaced D − F with correct versions of H177R, in which panel F overlays with the DIC image in E . The scale bar represents 15 μm. LLPS, liquid–liquid phase separation; PrP, prion protein; PrP C , cellular prion protein; TAMRA, 5(6)-carboxy-tetramethylrhodamine N -succinimidyl ester.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Purification, Variant Assay, Labeling, Fluorescence, Incubation, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Arg177 from the dog PrP strongly slows down the LLPS of full-length human PrP C , shifting the equilibrium phase boundary to a higher protein concentration. His177 from the human PrP, however, greatly enhances the LLPS of full-length dog PrP C , shifting the equilibrium phase boundary to a lower protein concentration. About 5 ( A , E , I , and M ), 10 ( B , F , J , and N ), 20 ( C , G , K , and O ), or 40 ( D , H , L , and P ) μM bacterial-purified full-length wildtype human PrP C (HoPrP-WT) ( A − D ) and its single variant H177R ( E − H ) as well as bacterial-purified full-length wildtype dog PrP C (CaPrP-WT) ( I − L ) and its single variant R177H ( M − P ) were labeled by TAMRA ( red fluorescence ) and incubated with 1× PBS (pH 7.4) containing 10% (w/v) PEG 8000 on ice to induce LLPS for 10 min. Liquid droplets of the human PrP C or the dog PrP C were observed by DIC confocal microscopy, with excitation at 561 nm. A − P , the merged images of the TAMRA images in Fig. S2 , A − P and the DIC microscopic images in Fig. S3 , A − P . The scale bar represents 10 μm. Q − T , the turbidity of PrP C condensates was measured at 600 nm and 25 °C. The dependence of turbidity changes for LLPS of HoPrP-WT ( Q ), its single variant H177R ( R ), CaPrP-WT ( S ), or its single variant R177H ( T ) on the concentration of HoPrP-WT ([HoPrP-WT]), H177R ([H177R]), CaPrP-WT ([CaPrP-WT]), or R177H ([R177H]) was expressed as mean ± SD (with error bars) of values obtained in three independent experiments. Representative calculation based on turbidity measurements to determine saturation concentration of the wildtype protein (HoPrP-WT or CaPrP-WT) ( open circle ) or the single variant (H177R or R177H) ( open square ). The orange and red lines are drawn through data points indicating the absence of LLPS, whereas the cyan and blue lines are drawn through data points in which robust LLPS of the wildtype protein (HoPrP-WT or CaPrP-WT) and the single variant (H177R or R177H) occurs, respectively. The concentration of protein at which these two lines intersect is an estimation of the saturation concentration. U and V , saturation concentrations of the wildtype proteins (HoPrP-WT and CaPrP-WT, blue ) and the single variants (H177R and R177H, red ) ( open black circles shown in scatter plots) were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. U , H177R, p = 0.00047; V , R177H, p = 0.0043. Statistical analyses were performed using the Student's t test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001 relative to control (the saturation concentration for wildtype PrP C ). DIC, differential interference contrast; LLPS, liquid–liquid phase separation; PrP, prion protein; PrP C , cellular prion protein; TAMRA, 5(6)-carboxy-tetramethylrhodamine N -succinimidyl ester.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Protein Concentration, Purification, Variant Assay, Labeling, Fluorescence, Incubation, Confocal Microscopy, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Arg177 from the dog PrP enhances fluorescence recovery and negatively modulates the LLPS of full-length human PrP C . His177 from the human PrP, however, decreases fluorescence recovery and positively modulates the LLPS of full-length dog PrP C . A − X , FRAP analysis on the selected liquid droplets of 50 μM bacterial-purified full-length wildtype human PrP C (HoPrP-WT) ( A − F ) and its single variant H177R ( G − L ) as well as bacterial-purified full-length wildtype dog PrP C (CaPrP-WT) ( M − R ) and its single variant R177H ( S − X ) labeled by TAMRA ( red fluorescence ) before (prebleach, A , G , M , and S ), during (0 s, B , H , N , and T ), and after photobleaching (10 s, C , I , O , and U ; 20 s, D , J , P , and V ; 30 s, E , K , Q , and W ; 150 s, F , L , R , and X ). The internal photobleaching is marked by a black square . Full-length PrP was incubated with 1× PBS (pH 7.4) containing 10% (w/v) PEG 8000 on ice to induce LLPS for 10 min, and liquid droplets were observed by confocal microscopy, with excitation at 561 nm. The scale bars represent 1 μm. The images in P , Q , and R almost did not change over time, and the images in U , V , W , and X almost did not change over time. Y and Z , normalized kinetics of fluorescence recovery data of the wildtype protein (HoPrP-WT or CaPrP-WT) ( open blue circle ) and the single variant (H177R or R177H) ( open red square ) obtained from FRAP intensity. The normalized fluorescence intensity is expressed as the mean ± SD of the values obtained in three independent experiments. The solid blue or red lines show the best single exponential fit for the fluorescence intensity–time curves. FRAP of phase-separated droplets of HoPrP-WT, its single variant H177R ( Y ), CaPrP-WT, or its single variant R177H ( Z ) revealed a fluorescence recovery rate of (8.42 ± 0.89) × 10 −2 s −1 , (9.27 ± 1.01) × 10 −2 s −1 , (6.94 ± 0.68) × 10 −2 s −1 , or (3.28 ± 0.09) × 10 −2 s −1 with a (25.9 ± 1.4)%, (58.2 ± 3.2)%, (28.8 ± 1.4)%, or (9.00 ± 0.11)% fluorescence recovery within 150 s. All FRAP experiments were repeated three times, and the results were reproducible. FRAP, fluorescence recovery after photobleaching; LLPS, liquid–liquid phase separation; PrP, prion protein; PrP C , cellular prion protein; TAMRA, 5(6)-carboxy-tetramethylrhodamine N -succinimidyl ester.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Fluorescence, Purification, Variant Assay, Labeling, Incubation, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Asp159 from the dog PrP slows down the LLPS of full-length human PrP C , shifting the equilibrium phase boundary to a higher protein concentration. Asn159 from the human PrP, however, enhances the LLPS of full-length dog PrP C , shifting the equilibrium phase boundary to a lower protein concentration. About 5 ( A and E ), 10 ( B and F ), 20 ( C and G ), or 40 ( D and H ) μM bacterial-purified single variant N159D of full-length human PrP C ( A − D ) and single variant D159N of full-length dog PrP C ( E − H ) were labeled by TAMRA ( red fluorescence ) and incubated with 1× PBS (pH 7.4) containing 10% (w/v) PEG 8000 on ice to induce LLPS for 10 min. Liquid droplets of the human PrP C or the dog PrP C were observed by DIC confocal microscopy, with excitation at 561 nm. We have replaced H with a correct version of D159N. The scale bar represents 10 μm. I and J , the turbidity of PrP C condensates was measured at 600 nm and 25 °C. The dependence of turbidity changes for LLPS of the single variant N159D of HoPrP-WT ( I ) or the single variant D159N of CaPrP-WT ( J ) on the concentration of N159D ([N159D]) or D159N ([D159N]) was expressed as mean ± SD (with error bars) of values obtained in three independent experiments. Representative calculation based on turbidity measurements to determine saturation concentration of the single variant (N159D or D159N) ( open square ). The red line is drawn through data points indicating the absence of LLPS, whereas the blue line is drawn through data points in which robust LLPS of the single variant (N159D or D159N) occurs. The concentration of protein at which these two lines intersect is an estimation of the saturation concentration. K and L , saturation concentrations of the wildtype proteins (HoPrP-WT and CaPrP-WT, blue ) and the single variants (N159D and D159N, red ) ( open black circles shown in scatter plots) were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. K , N159D, p = 0.0030; L , D159N, p = 0.0017. Statistical analyses were performed using the Student's t test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001 relative to control (the saturation concentration for wildtype PrP C ). M − V , FRAP analysis on the selected liquid droplets of 50 μM bacterial-purified single variant N159D of human PrP C ( M − Q ) and single variant D159N of dog PrP C ( R − V ) labeled by TAMRA ( red fluorescence ) before (prebleach, M and R ), during (0 s, N and S ), and after photobleaching (10 s, O and T ; 30 s, P and U ; 150 s, Q and V ). The internal photobleaching is marked by a black square . The scale bars represent 1 μm. The images in O , P , and Q almost did not change over time, and the images in T , U , and V almost did not change over time. W , normalized kinetics of fluorescence recovery data of D159N ( open blue circle ) and N159D ( open red square ) obtained from FRAP intensity. The normalized fluorescence intensity is expressed as the mean ± SD of the values obtained in three independent experiments. The solid blue lines or red lines show the best single exponential fit for the fluorescence intensity–time curves. FRAP of phase-separated droplets of single variant N159D of human PrP C or single variant D159N of dog PrP C revealed a fluorescence recovery rate of (8.70 ± 0.36) × 10 −2 s −1 or (7.03 ± 0.34) × 10 −2 s −1 with a (45.2 ± 1.0)% or (23.8 ± 0.6)% fluorescence recovery within 150 s. All FRAP experiments were repeated three times, and the results were reproducible. DIC, differential inference contrast; FRAP, fluorescence recovery after photobleaching; LLPS, liquid–liquid phase separation; PrP, prior protein; PrP C , cellular prion protein; TAMRA, 5(6)-carboxy-tetramethylrhodamine N -succinimidyl ester.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Protein Concentration, Purification, Variant Assay, Labeling, Fluorescence, Incubation, Confocal Microscopy, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Arg177 from the dog PrP significantly inhibits amyloid formation of human PrP, but His177 from the human PrP greatly enhances fibril formation of dog PrP. Samples (20 μM) of HoPrP-WT ( blue ) and its single variant H177R ( red ) ( A − C ) as well as CaPrP-WT ( blue ) and its single variant R177H ( red ) ( D − F ) were incubated in 1× PBS (pH 7.4) containing 2 M GdnHCl with agitation at 220 rpm and then analyzed by thioflavin T (ThT) binding assay ( A , B , D , and E ) and 8-anilino-1-naphthalene-sulfonic acid (ANS) binding assay ( C and F ). A , C , D , and F , the ThT and ANS fluorescence intensities were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. The solid lines show the best exponential fit for the ThT/ANS intensity–time curves. B and E , the fibril formation lag time in the wildtype proteins (HoPrP-WT and CaPrP-WT, blue ) and the single variants (H177R and R177H, red ) ( open black circles shown in scatter plots) was determined by fitting ThT fluorescence intensity versus time to a sigmoidal equation and was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. B , H177R, p = 0.000034; E , R177H, p = 0.0012. Statistical analyses were performed using the Student's t test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001 relative to control (the lag time for wildtype PrP) ( B and E ). ANS, 8-anilino-1-naphthalene-sulfonic acid; GdnHCl, guanidine hydrochloride; PrP, prion protein; ThT, thioflavin T.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Variant Assay, Incubation, Binding Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Arg177 and Asp159 from dog prion protein slow liquid–liquid phase separation and inhibit amyloid formation of human prion protein

doi: 10.1016/j.jbc.2023.105329

Figure Lengend Snippet: Arg177 from the dog PrP strongly slows down the LLPS of GPI-anchored and glycosylated full-length human PrP C , shifting the equilibrium phase boundary to a higher protein concentration. Full-length wildtype human PrP C and its single variant H177R were expressed and purified in sf9 insect cells. A and B , identification of N-linked glycosylation sites in the insect cell–purified HoPrP-WT using mass spectrometry (MS). The Coomassie blue–stained gels of SDS-PAGE of GPI-anchored and glycosylated HoPrP-WT were scissored out, chopped, trypsinized, and then analyzed with nano-LC–MS/MS. A , an MS 2 analysis of parent peptide V 176 HDCVNITIK 185 digested by trypsin. Analysis of the y-ions ( y 5 + 1 -NH 3 and y 4 + 1 ) indicates +1 Da mass shift for an Asn-to-Asp conversion at position 181 (572.36 plus 17.026 minus 474.37 = 115.02 Da, 115.02 minus 114.04 = 0.98 Da), demonstrating N-linked glycosylation at Asn181 in the insect cell–purified HoPrP-WT. B , an MS 2 analysis of parent peptide G 195 ENFTETDVK 204 digested by trypsin. Analysis of the y-ions ( y 8 + 1 and y 7 + 1 ) also indicates +1 Da mass shift for an Asn-to-Asp conversion at position 197 (954.47 minus 839.43 = 115.04 Da, 115.04 minus 114.04 = 1.00 Da), demonstrating N-linked glycosylation at Asn197 in the insect cell–purified HoPrP-WT. C − J , 10 ( C ), 20 ( D ), 30 ( E ), 40 ( F and G ), 50 ( H ), 60 ( I ), or 70 ( J ) μM post-translationally modified wildtype human PrP C (HoPrP-WT) ( C − F ) and H177R PrP C ( G − J ) were labeled by TAMRA ( red fluorescence ) and incubated with 1× PBS (pH 7.4) containing 12.5% (w/v) PEG 8000 on ice to induce LLPS for 10 min. Liquid droplets of the human PrP C were observed by confocal microscopy, with excitation at 561 nm. C − J , the merged images of the TAMRA images in Fig. S8 , A − D and I − L , and the brightfield images in Fig. S8 , E − H and M − P . The scale bar represents 10 μm. K and L , the turbidity of PrP C condensates was measured at 600 nm and 25 °C. The dependence of turbidity changes for LLPS of GPI-anchored and glycosylated HoPrP-WT ( K ) or its single variant H177R ( L ) on the concentration of HoPrP-WT ([HoPrP-WT]) or H177R ([H177R]) was expressed as mean ± SD (with error bars) of values obtained in three independent experiments. Representative calculation based on turbidity measurements to determine saturation concentration of HoPrP-WT ( open circle ) or H177R ( open square ). The orange and red lines are drawn through data points indicating the absence of LLPS, whereas the cyan and blue lines are drawn through data points in which robust LLPS of HoPrP-WT and H177R occurs, respectively. The concentration of protein at which these two lines intersect is an estimation of the saturation concentration. M , saturation concentrations of HoPrP-WT ( blue ) and H177R ( red ) ( open black circles shown in scatter plots) were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. H177R, p = 0.0032. Statistical analyses were performed using the Student's t test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001 relative to control (the saturation concentration for wildtype PrP C ). GPI, glycosylphosphatidylinositol; LLPS, liquid–liquid phase separation; PrP, prior protein; PrP C , cellular prion protein; TAMRA, 5(6)-carboxy-tetramethylrhodamine N -succinimidyl ester.

Article Snippet: The recombinant full-length wildtype human PrP and its variant H177R were purified by the method of size-exclusion chromatography using an ÄKTA Pure instrument (GE Healthcare).

Techniques: Protein Concentration, Variant Assay, Purification, Mass Spectrometry, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Modification, Labeling, Fluorescence, Incubation, Confocal Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes

doi: 10.1101/2020.01.15.907444

Figure Lengend Snippet: (A) Representative time-lapse images showing breakage of the connection between the growing tip of a newly-formed ER tubule (magenta) and a lysosome (green) in EGFP-VAPA(KD/MD)-expressing cells. See table S2 for the compromised contact sites upon VAPA(KD/MD) overexpression. See Movie S5. (B) Average velocities (black spots) of initially ER-tethered lysosomes that become detached (red arrow) from their associated ER growing tips. Detachment events are ER-lysosome connection breakages in EGFP-VAPA(KD/MD)-expressing cells as in (A). **= p<0.01, **** = p<0.0001 (Tukey’s one-way ANOVA). Velocities of 22 events from three independent experiments were quantified. See table S3. (C) Left: Diagram depicting the regions defined as perinuclear and peripheral regions for the following quantification of lysosome distribution, same definition for ( E ). Right: Percentage of the ER comprising tubules upon knockdown of lysosome motion adaptors. Data are shown as ± SEM. **** = p<0.0001 (Tukey’s one-way ANOVA). Data from 20 cells from 3 independent experiments were analyzed for each condition. See table S4. (D) Representative images showing the distribution of lysosomes and ER tubules in control cells and in cells treated with siRNAs. (E) i : Diagram depicting individual components of the chemogenetic system. ii : Quantification of lysosome intensity change and percentage of the ER comprising tubules after 1 hr inducer treatment. Data are shown as ± SEM. **** = p<0.0001 (Student’s t test). For lysosome intensity analysis, N = 10, for ER tubule percentage, N = 20. iii : Representative images showing the distribution of lysosomes and ER tubules in control cells and in cells treated with inducers. (F) Optogenetic assay for repositioning of LAMP1. ( G-J ): Live-cell imaging ( G ), representative zoom-ins ( H ), representative kymograph ( I ) and quantification ( J ) of LAMP1-mCherry-iLID and YFP-SEC61B in COS-7 cells expressing opto-kinesin before or during activation. White arrows indicate lysosome pulling ER tubule (yellow asterisk). Quantification shows mean (± S.E.M.) normalized peripheral SEC61 and LAMP1 intensity of 8 cells. Blue box indicates illumination with blue light. Scale bars represent 1 μm in ( A ) ( G ) ( H ) and ( I ), 5 μm in ( D ) and ( E ). s, seconds.

Article Snippet: Full length wildtype human LAMP1, was derived from LAMP1-mGFP , a gift from Esteban Dell’Angelica (University of California, Los Angeles, CA; Addgene plasmid #34831).

Techniques: Expressing, Over Expression, Knockdown, Control, Live Cell Imaging, Activation Assay