Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Interaction of GCase variants with their lysosomal transporter LIMP‐2. A) Crystal structure of GCase (PDB: 5LVX). [ ⁴⁴ ] The domains and motifs are colored as follows: pink: antiparallel beta‐sheet (domain 1); grey: TIM barrel (domain 2) containing active site (green); blue: beta‐barrel (domain 3); [ ⁴⁵ ] orange: proposed LIMP‐2‐binding motif. [ ⁴² ] Single amino acids comprising the active site, as well as amino acids exchanged in common disease‐associated GCase variants E326K, N370S, and L444P are labeled and highlighted. B) GCase activity in cell lysates of HEK 293T cells overexpressing GCase variants (n = 3‐8; mock: 8; wt: 8; E326K: 5; N370S: 3; L444P: 5; individual transfections). All disease‐associated variants show significantly reduced activity. E326K shows a residual activity of 40.0 ± 2.2%, N370S, and L444P do not differ significantly from the mock. C) Illustration of the lysosomal transport of GCase and its interaction with LIMP‐2. GCase binds to LIMP‐2 in the ER to form a transporting complex. After trafficking through the Golgi, the LIMP‐2/GCase complex is sorted to lysosomal compartments where low pH triggers complex dissociation. D) Western blot analyses of whole‐cell lysates and LE fractions of HEK 293T cells expressing GCase variants and FL‐LIMP‐2 or FL‐LIMP‐2‐3xD as a control. LE fractions are higher in GCase, LIMP‐2, and the lysosomal protein LAMP‐2A. Enriched fractions of cells overexpressing FL‐LIMP‐2 show increased levels of GCase. Blots containing a dashed line were spliced for a more comprehensive data presentation (both parts are from the same image). E) Quantification of western blot signal from lysate and LE HEK 293T samples (Figure ) (n = 3‐6; mock: 6; wt: 6; E326K: 3; N370S: 3; L444P: 3; individual cell harvests). The abundance of wt, E326K, and N370S GCase was significantly increased in LE fractions when FL‐LIMP‐2 was co‐expressed compared to the co‐expression of the non‐binding 3xD control. In general, the GCase signal was increased in LE fractions but did not reach statistical significance for FL‐LIMP‐2‐3xD samples and FL‐LIMP‐2 + L444P. F) GCase activity in cell lysates and LE fractions of HEK 293T cells overexpressing GCase variants and FL‐LIMP‐2 or FL‐LIMP‐2‐3xD (n = 3‐7; mock: 7; wt: 7; E326K: 4; N370S: 3; L444P: 4; individual cell harvests). Overexpression of FL‐LIMP‐2 resulted in an increase of GCase activity in the lysate itself, but also in the LE fraction for wt GCase and the E326K variant, while no significant differences were observed for the N370S and L444P variant. Asterisks (*) indicate significant differences between cell lysate and enriched fractions. Pound signs (#) indicate significant differences in relation to the respective 3xD control sample (example: wt + FL‐LIMP‐2 lysate versus wt + FL‐LIMP‐2‐3xD lysate). Statistics: replicates (dots) with a mean (column) ± SEM (B,E,F). Tests: One‐way ANOVA with Tukey's multiple comparison test (B); Two‐way ANOVA with Tukey's multiple comparison test (E,F). * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001; ## p < 0.01, #### p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Binding Assay, Labeling, Activity Assay, Transfection, Western Blot, Expressing, Control, Over Expression, Variant Assay, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Characterization of primary human fibroblasts of controls and patients with PD and GD. A) Western blot of whole cell lysates from control (CTRL‐1,2,3), PD patient‐derived (PD‐1,2), and GD patient‐derived (GD) primary human fibroblasts. Quantitative analysis of signals can be found in Figure (Supporting Information). GCase levels were comparable between control and PD but diminished in GD (Figure , Supporting Information). LIMP‐2 levels were increased in PD compared to controls (Figure , Supporting Information). LAMP‐2A and calnexin levels varied between cell lines and did not show any significant trend between groups (Figure , Supporting Information). GAPDH and CBB staining of the gel are presented as loading controls. B) GCase activity in whole‐cell lysates of control, PD, and GD fibroblast cell lines (n = 3‐7; CTRL‐1,2,3: 7; PD‐1,2: 7; GD: 3; individual cell harvests). The E326K lines PD‐1 and PD‐2 showed significantly lower GCase activity (66.41±2.69% and 73.56±6.50%) compared to the control lines. In contrast, activity in the GD line (GBA1 L444P/L444P ) was almost fully abolished (3.11±0.11% residual activity). C) Colocalization of LIMP‐2 and GCase in primary human fibroblasts determined via Pearson's correlation coefficient (n = CTRL‐1: 30; CTRL‐2: 38; CTRL‐3: 33; PD‐1: 44; PD‐2: 51; GD: 27; individual cells from multiple images). PD patient fibroblasts PD‐1 and PD‐2 show mildly reduced colocalization of LIMP‐2 and GCase compared to control lines. In the GD patient line, colocalization was diminished even further. D) Representative immunofluorescence images of primary human fibroblast lines. Objective magnification: 40x. Green: LIMP‐2; red: GCase, blue: DAPI. All lines show the vesicular distribution of LIMP‐2, indicating lysosomes. Control and PD lines show visible colocalization of GCase with the LIMP‐2 signal. In GD fibroblasts, the GCase signal was less intense and less granular, representing lower expression and lysosomal localization. Statistics: replicates (dots) with mean (column) ± SEM (B); violin plot with median (dashed line) and quartiles (dotted line) (C). Tests: Nested one‐way ANOVA with Tukey's multiple comparison test (B,C). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Western Blot, Control, Derivative Assay, Staining, Activity Assay, Immunofluorescence, Expressing, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Purification of GCase utilizing soluble (s) LIMP‐2. A) Workflow for the purification of sLIMP‐2/GCase complex from HEK 293F cells. A culture is transfected to co‐express sLIMP‐2 and GCase. After 96 h, the conditioned medium is collected and Ni‐NTA purification of His‐tagged proteins is performed, yielding sLIMP‐2 and sLIMP‐2/GCase complex. Elution fractions are concentrated and separated via SEC, yielding purified sLIMP‐2/GCase protein complex. B) Cartoon of intracellular mechanisms of interaction and secretion of GCase with the soluble sLIMP‐2 construct. A soluble sLIMP‐2/GCase complex is formed in the ER. The IgK leader sequence on sLIMP‐2 then facilitates secretion of the LIMP‐2/GCase complex instead of sorting GCase to the lysosome. In comparison, the sLIMP‐2‐3xD control construct does not bind GCase and therefore does not facilitate secretion of GCase. C) GCase activity in the supernatant of HEK 293F cells overexpressing wt GCase along either sLIMP‐2 or sLIMP‐2‐3xD (n = 3; individual transfections). Samples were taken 0‐72 h after transfection. In co‐expression with sLIMP‐2, GCase activity is higher and stable over the course of the measurements, whereas activity in the control is lower and diminishes over time. The statistical significance indicated (*) represents a comparison between both sample groups at a given time point. D) Representative western blot of analytical samples from Ni‐NTA purification of the sLIMP‐2/GCase complex from HEK 293F supernatant. Parts of the blot are shown with increased contrast to visualize faint signals. Splicing is indicated by a dashed line. Abbreviations: cond. SN: conditioned supernatant; dep. SN: depleted supernatant; wash 1/4: flow‐through of washing steps 1/4 (4 total); elution F1: elution fraction 1 (5 total). Elution of protein from Ni‐NTA resin yielded sLIMP‐2 and GCase. E) Representative SEC profiles of samples from Ni‐NTA purification of sLIMP‐2/GCase (blue), sLIMP‐2 (black), and His‐tagged GCase (grey, dashed) using a Superdex 200 Increase 3.2/300 column. sLIMP‐2 and sLIMP‐2/GCase samples share a peak at fraction 8 corresponding to the sLIMP‐2 monomer. An additional peak in fraction 7 is visible in the sLIMP‐2/GCase sample, corresponding to the sLIMP‐2/GCase complex. His‐tagged GCase runs as a major peak in fractions 4/5 with training smaller peaks in later fractions. F) Western Blot analyses of SEC fractions (corresponding to Figure ). Fractions of the sLIMP‐2/GCase sample show strong GCase and LIMP‐2 signals with maxima at fractions 7/8 and 8/9 respectively. Fractions of sLIMP‐2 show the same distribution with a weak GCase signal. His‐tagged GCase was most abundant in fractions 5/6. Statistics: replicates (dots, squares) with mean (line) ± SEM (C). Tests: Two‐way ANOVA with Sidak's multiple comparison test (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001 .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Purification, Transfection, Construct, Sequencing, Comparison, Control, Activity Assay, Expressing, Western Blot

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Design and effect of LIMP‐2‐derived helix 5 peptide on GCase function. A) Sequence of custom LIMP‐2 peptides. The peptides comprise the wt or 3xD variant of helix 5 of LIMP‐2 (orange), flanked by an N‐terminal lysosomal KFERQ sequence for lysosomal targeting (green) and a C‐terminal TAT‐peptide for cell penetration (pink). Linker regions are indicated in grey. B) Cartoon of uptake of LIMP‐2‐derived peptide into cells and the lysosome, where the peptide interacts with GCase, boosting its lysosomal function. C,D) Interaction of L2H5‐wt and GCase‐His at cytosolic (C) and lysosomal (D) pH as determined by MST (n = 3 sample preparations per condition). Dots represent mean ± SEM. With increasing concentration of ligand (L2H5‐wt), changes in the MST signal (FNorm) could be observed at both conditions, indicating binding to GCase. Fitting of a Kd model (red line) yielded estimated affinities in the nanomolar range for pH 7.4 and micromolar range for pH 5.0. At pH 7.4, higher L2H5 concentrations lead to a second change in the MST signal, hinting toward a second binding event with lower affinity (illustrated as a grey dashed line). Grey datapoints were disregarded for the Kd model fit (red line). E) Enzyme activity of Cerezyme in the presence of varying concentrations of L2H5‐wt or −3xD peptides (n = 3; individual experiments). An activating effect of L2H5‐wt was first observed in the micromolar range and increased further with peptide concentration. The addition of 10 µM of L2H5 led to a 2.63 ± 0.22‐fold increase of GCase activity. At peptide concentrations above 20 µM, precipitation of the peptide occurred as indicated by a dashed grey line. F) Effect of L2H5 peptides on the activity of recombinant GCase in conditioned HEK 293F media after overexpression of GCase variants (n = 3, individual experiments). The activity of wt GCase and E326K were increased in the presence of 10 µM L2H5‐wt. The activity of N370S and L444P were unaffected. Statistics: mean (dot) ± SEM (C,D), replicates (dots, squares) with mean (line) ± SEM (E); Mean (column) ± SEM (F). Tests: non‐linear regression Kd model (C,D); two‐way ANOVA with Tukey's multiple comparison test (F). * p < 0.05, **** p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Derivative Assay, Sequencing, Variant Assay, Concentration Assay, Binding Assay, Activity Assay, Recombinant, Over Expression, Comparison

Journal: Advanced Science

Article Title: Activation and Purification of ß ‐Glucocerebrosidase by Exploiting its Transporter LIMP‐2 – Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease

doi: 10.1002/advs.202401641

Figure Lengend Snippet: Uptake and effect of L2H5 peptides on control and PD fibroblasts. A) Assessment of cell death via LDH activity in culture medium in CTRL‐2, PD‐1, and PD‐2 fibroblast lines (n = 3; wells from the 96‐well plate). Cells were treated with varying concentrations of L2H5‐wt peptide for 72 h. concentrations up to 10 µM did not lead to an increase in cell death. At a concentration of 20 µM however, LDH activity in the medium was significantly increased, indicating increased cell death due to treatment. Effects were comparable between all three lines. Significance is shown in comparison to the 0 µM data group for each cell line respectively. B) Presence of tryptic exogenous L2H5‐wt and endogenous LIMP‐2‐derived peptides in LE fractions of HEK293T cells after treatment with PBS (neg. ctrl.) or 5 µM L2H5‐wt for 2 h and 24 h. Determined via mass spectrometry. The positive control represents a sample spiked with 0.5 µg of L2H5 before analysis. L2H5‐wt‐specific peptides 1‐4 were detected in the pos. ctrl (all 4) and the cells treated with L2H5‐wt for 2 h (peptides 2 and 3). Low amounts of peptides 2 and 3 were also detected at the 24 h time point, but only by matching (indicated with an asterisk) and only in two of the three samples. Peptide 5, which is a tryptic product of both L2H5‐wt and endogenous LIMP‐2, was detected in all samples as expected, with higher abundance in the spiked control and the 2 h treated samples. C) Representative immunofluorescence image of CTRL‐2 fibroblasts with GFP‐labeled lysosomes (CellLight Lysosomes‐GFP) after 2 h of treatment with 0.25 µg µL −1 FRed‐L2H5‐wt. Objective magnification: 63x. Top: single channels. Middle: merged picture. Bottom: zoomed in single channels and merged picture of area inside a white frame. Green: GFP; red: FusionRed; blue: DAPI. FusionRed signal dots were visible within the cell. Some dots were surrounded by GFP signal located in the lysosomal membrane, thus confirming the presence of FRed‐L2H5‐wt inside the lysosome as shown by white arrows and in the zoomed‐in section. D) Live cell GCase activity in primary human control fibroblasts and PD‐patient‐derived fibroblasts harboring E326K mutations (n = 3; wells of a 96‐well plate). The cells were treated with PBS or L2H5 peptides (wt and 3xD). The graph shows lysosomal GCase activity as an area between curves (see materials and methods). In all three cell lines, lysosomal GCase activity was dramatically boosted after treatment with L2H5‐wt. In contrast, treatment with the non‐binding L2H5‐3xD peptide did not affect lysosomal GCase activity. See Figure (Supporting Information) for individual activity graphs with replicates. Statistics: replicates (dots) with mean (column) ± SEM (A); mean (column) ± SEM (D). Tests: Two‐way ANOVA with Dunnett's multiple comparison test (A); Two‐way ANOVA with Tukey's multiple comparison test (D). * p < 0.05, *** p < 0.001 **** p < 0.0001, n.s.: not significant .

Article Snippet: For expression of full‐length human LIMP‐2 (Sino Biological Inc., Peking, China, #HG11063‐CH), and untagged human GCase wt (Sino Biological Inc., Peking, China, #HG12038‐UT), commercially available expression constructs were used.

Techniques: Control, Activity Assay, Concentration Assay, Comparison, Derivative Assay, Mass Spectrometry, Positive Control, Immunofluorescence, Labeling, Membrane, Binding Assay

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 enhances SCARB2 expression and EV-A71 attachment to cells. (a) TRIB3 depletion decreased the protein level of SCARB2 . Tandem Mass Tag™-LC-MS/MS analysis evaluated the protein level of SCARB2 in WT cells and TRIB3-KO cells ( n = 2). (b, c, g, i) TRIB3 depletion decreased the protein level of SCARB2. SCARB2 expression were detected with WB (b, n = 3), qRT-PCR assay (c, n = 4), Immunofluorescence (g) and flow cytometry (i, n = 3) in WT cells and TRIB3-KO cells. (d, e, f, h) TRIB3 overexpression increased the protein level of SCARB2. HCT-8 cells were treated with Control-HA or TRIB3-HA plasmids. At 24 h after transfection, cells were harvested for WB assay with indicated antibodies (d, n = 3), qRT-PCR assay (e, n = 4), Immunofluorescence (f) and flow cytometry (h, n = 3). Percentage of SCARB2 positive cells was calculated with FCS express software. (j, k) TRIB3 overexpression promoted EV-A71 binding and TRIB3 knockout decreased EV-A71 binding. HCT-8 cells were transfected with indicated plasmids. At 24 h after transfection, HCT-8 cells were rested at 4°C for 1 h and infected with EV-A71 (MOI = 1.0) on ice for 30 min. The cells were rinsed with PBS and harvested. Cell-associated EV-A71 RNA were measured by a qRT-PCR assay (j. n = 3). WT cells and TRIB3-KO cells were rested at 4°C for 1 h and then infected with EV-A71 (MOI = 1.0) on ice for 30 min. The cells were rinsed with PBS and harvested. Cell-associated EV-A71 RNA were measured by were a qRT-PCR assay (k, n = 3). P < 0.05 , Student’s t -test (b, c, d, e, h, i, j, k).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR, Immunofluorescence, Flow Cytometry, Over Expression, Transfection, Software, Binding Assay, Knock-Out, Infection

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 represses ubiquitylation and degradation of SCARB2. (a) 293 T cells were co-transfected with a plasmid expressing SCARB2-Myc and TRIB3-HA or a vector plasmid. At 24 h post transfection, cells were incubated with cycloheximide (CHX) (10 μg/ml) for indicated times. Proteins were detected by WB with the indicated antibodies. P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test. (b) The effect of TRIB3 overexpression on SCARB2 ubiquitylation in vitro . 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-Myc Ab. The ubiquitylated SCARB2 was detected with WB assay. (c, d) 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-Myc Ab or anti-Flag Ab. The ubiquitylated SCARB2 was detected with WB assay. (e) The KDC region of TRIB3 was responsible for its promoting EV-A71 infection. (Upper panel) Schematic diagram of TRIB3 deletion mutants. (Lower panel) Cell extracts from 293 T cells transfected with the indicated plasmids and infected with EV-A71 were resolved by SDS-PAGE, proteins were detected by WB with the indicated antibodies. (f) Cell extracts from TRIB3-KO cells transfected with the indicated plasmids and infected with EV-A71 were resolved by SDS-PAGE, proteins were detected by WB with the indicated antibodies. (g) Effect of KDC deletion in TRIB3 on SCARB2 ubiquitination in 293 T cells. (h) Cell extracts from 293 T cells transfected with the indicated plasmids and infected with EV-A71 were detected by WB with the indicated antibodies. (i) The relationship between TRIB3 and SCARB2. 293 T cells were transfected with indicated plasmids and cell extracts were IP with anti-HA or anti-Flag Ab. (j) The relationship between endogenous TRIB3 and SCARB2. HCT-8 cells extracts were IP with anti-TRIB3.

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Over Expression, In Vitro, Infection, SDS Page

Journal: Emerging Microbes & Infections

Article Title: Tribbles pseudokinase 3 promotes enterovirus A71 infection via dual mechanisms

doi: 10.1080/22221751.2024.2307514

Figure Lengend Snippet: TRIB3 facilitates EV-A71 replication in a SCARB2-independent manner. (a) HCT-8 cells were transfected with control-HA or TRIB3-HA plasmids. At 3 h or 10 h post transfection, HCT-8 cells were harvested for WB assay with indicated antibodies. (b) HCT-8 cells were transfected with control-HA or TRIB3-HA plasmids. At 3 h post transfection, HCT-8 cells were mock-infected or infected with EV-A71 (MOI = 1.0) for 7 h. The cells were harvested for WB assay with indicated antibodies. (c) 293 T cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h post transfection, the cells were transfected again with EV-A71 subgenomic replicon RNA and luciferase reporter activities were determined at different time ( n = 6). (d) HCCLM3 cells and SCARB2-KO HCCLM3 cells were harvested for WB assay with indicated antibodies. (e) SCARB2-KO HCCLM3 cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h post transfection, the cells were transfected again with EV-A71 subgenomic replicon RNA and luciferase reporter activities were determined at different time ( n = 6). (f–g) SCARB2-KO HCCLM3 cells were transfected with control-HA or TRIB3-HA plasmids. At 24 h after transfection, cells were mock-infected or infected with EV-A71 (MOI = 1) for 24 h. The cells were harvested for WB assay with indicated antibodies (f, n = 3) and qRT-PCR assay (g, n = 3). P < 0.05, two-way ANOVA with Holm-Sidak multiple comparisons test (c, e) or Student’s t -test (f, g).

Article Snippet: TRIB3-Myc (HG10731-CM), SCARB2-Flag (HG11063-CF), SCARB2-Myc (HG11063-CM) and NEDD8-Myc (HG15620-CM) were purchased from Sino Biological Inc (Beijing, China).

Techniques: Transfection, Infection, Luciferase, Quantitative RT-PCR