gfp rab8a  (Addgene inc)

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW and LRRK2 KW ; (B) and (C) Close up of the inhibitor binding pocket from cryo-EM maps and models of LRRK2 RCKW bound to the type-I inhibitor MLi-2 (PDB: 8TXZ) (B) and type-II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange: C-lobe; light orange: N-lobe; black: DYG motif; grey: G-loop; green: activation loop. (D) Scheme depicting our hybrid design strategy to develop potent and selective type-II inhibitors for LRRK2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Construct, Binding Assay, Cryo-EM Sample Prep, Activation Assay

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) The co-crystal structure of RN129 (28) with CLK3 highlighting the type-II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3); (B) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI); (C) and ( D) Close ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 ( C ) and LRRK2 RCKW :GZD824 (PDB: 8TZE) ( D ).

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue. The 18.5 K ΔTm shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size correlates with the degree of ΔTm shift, as indicated in the legend; (B) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20 K ΔTm shift of LRRK2 KW is highlighted in red. The bubble size for each kinase correlates with the ΔTm shifts, as indicated in the legend (as in A ). Kinases with ΔTm > 6 K are labeled; (C) Waterfall plots of the ReactionBiology 33 PanQinase™ screen of RN341 at 1 µM and 10 µM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to < 22 % of the control value are labeled; (D) Off-target validation from both screens via in cellulo nanoBRET assay in 2 biological replicates, error bars ± sd, EC 50 (JNK2) = 2.7 µM, EC 50 (STK10) = 1.5 µM, EC 50 (MAPK14) = 1.7 µM, EC 50 (TTK) = 3.2 µM, EC 50 (CDKL1) = 17 µM, EC 50 (CLK1) = 6.0 µM, EC 50 (JNK3) = 15 µM, EC 50 (DYRK2) = >20 µM, EC 50 (SLK) >20 µM, EC 50 (DDR2) >20 µM, EC 50 (STK17B) = >20 µM.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) and (B) Dose response curve of RN277 ( 30 ) and RN341 ( 32 ) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a vs. non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341; (C) Western blots from 293T cells transiently co-transfected with LRRK2 (full-length) and GFP-Rab8a for 48h prior to treatment with a dilution series of RN277 ( 30 ) and RN341 ( 32 ) for 4h. DMSO and MLi-2 (500 nM) treatment for 4h were used as negative and positive controls, respectively. Lysed cells were immunoblotted for LRRK2, total GFP-Rab8a, phospho-Rab8a (pT72) and GAPDH as a loading control; (D) Quantification from four independent western blots showing the ratio of GFP-pRab8a to total GFP-Rab8a upon treatment with RN277 ( 30 ) and RN341 ( 32 ) at the indicated concentrations. Statistical analysis was performed using one-way ANOVA analysis with Tukey’s multiple comparison of means. ****p<0.0001, error bars ± s.e.m. (E) Western blots from 293T cells transiently co-transfected with LRRK2 (full-length) and GFP-Rab8a, treated with DMSO (control), 500 nM MLi-2, 5 µM RN277 or 5 µM RN341 for 4h, 48h post-transfection. Lysed cells were immunoblotted for LRRK2, phospho-LRRK2 (pS935), total GFP-Rab8a, phospho-Rab8a (pT72) and GAPDH as a loading control; (F) Quantification from four independent western blots showing the ratio of GFP-pRab8a to total GFP-Rab8a upon treatment with the indicated inhibitors (as in D ). Statistical analysis was performed using a one-way ANOVA analysis with Tukey’s multiple comparison of means. ****p<0.0001, error bars ± s.e.m.; (G) Quantification from four independent western blots showing the ratio of pLRRK2 to total LRRK2 upon treatment with the indicated inhibitors. Statistical analysis was performed using a one-way ANOVA analysis with Tukey’s multiple comparison of means. *p 0.0469, error bars ± s.e.m.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection, Control, Comparison

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Schematic of the single-molecule in vitro motility assay; (B) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type-I inhibitor MLi-2 (5 µM) in the presence or absence of LRRK2 RCKW . Scale bars 5 µM (x) and 30 s (y); (C) Quantification of the percentage (mean ± s.e.m) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 µM). Three technical replicates were collected per condition, with data points represented as circles, triangles and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; DMSO condition ***p 0.0007, MLi-2 condition ***p 0.0003, One-way ANOVA with Holm-Sidaks multiple comparison test within drug only; (D) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type-II inhibitors Ponatinib, RN277 and RN341 (5 µM) in the presence or absence of LRRK2 RCKW . Scale bars 5 µM (x) and 30 s (y); (E) Quantification of the percentage (mean ± s.e.m) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type-II inhibitors Ponatinib, RN277 and RN341 (5 µM). Three technical replicates were collected per condition, with data points represented as circles, triangles and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; ***p 0.0003, One-way ANOVA with Holm-Sid-aks multiple comparison test within drug only.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: In Vitro, Motility Assay, Concentration Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2

doi: 10.1016/j.jbc.2024.107469

Figure Lengend Snippet: DARPin E11 binds to LRRK2 RCKW with high affinity . A , domain architecture of the LRRK2 protein. The same color scheme is used in all figures. The first residue of each domain is indicated. B and C , surface plasmon resonance analysis of DARPin E11 binding to LRRK2 RCKW . Recombinant LRRK2 RCKW was immobilized on a sensor chip and varying concentrations of DARPin E11 were added in the mobile phase. Sensograms are shown (no fit or errors were applied) ( B ) and the plateau values from the dose-response curves ( C ) are shown and fit to the Langmuir equation according to the least square method. A dissociation constant (K D ) of 70 nM ± 11 nM (SEM) was determined. D , the LRRK2 RCKW :E11 complex was subjected to size exclusion chromatography (SEC). Elution fractions were analyzed by SDS PAGE and visualized by Coomassie staining. The co-elution of LRRK2 RCKW and DARPin E11 showed that the complexes were stable during the SEC run. Molecular weight markers are noted on the left and the molecular weights of LRRK2 RCKW and DARPin E11 on the right. E , analysis of the LRRK2 RCKW :E11 complex by mass photometry. The molecular weight obtained from the yellow peak corresponds to that of a 1:1 LRRK2 RCKW :E11 complex. The tabular data for this figure can be found at: https://doi.org/10.5281/zenodo.10471514 . DARPin, designed ankyrin-repeat protein; LRRK2, leucine rich repeat kinase 2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of WT, full-length, untagged LRRK2 (pCDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene; RRID: Addgene_49543) or 1000 ng LRRK2, 500 ng GFP-Rab8A, and 500 ng of 8xHis-DARPin E11-3xFLAG using polyethylenimine.

Techniques: Residue, SPR Assay, Binding Assay, Recombinant, Size-exclusion Chromatography, SDS Page, Staining, Co-Elution Assay, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2

doi: 10.1016/j.jbc.2024.107469

Figure Lengend Snippet: Cryo-EM structure of the LRRK2 RCKW :DARPin E11 complex. A , Cryo-EM map ( left ), model ( middle ), and schematic representation ( right ) of LRRK2 RCKW bound to DARPin E11. Because of the focused refinement strategy used to maximize the resolution of the DARPin-WD40 part of the structure, only the kinase C-lobe and the WD40 domain are seen in this map. E11 binds to one of the faces of the WD40 domain, opposite the kinase active site, next to the central WD40 cavity. The eyes and arrows surrounding the rectangle on the model indicate the direction of the views shown in panels ( C – E ). B , surface representation of hydrophobicity of LRRK2 KW bound to DARPin E11, displaying the latter as a model. The most polar residues are shown in blue , and the most hydrophobic residues are shown in gold . C – E , close-up views of the binding interface. Key residues for the interaction are highlighted. F and G , Cryo-EM maps, FSC plots, and model-to-map fits for LRRK2 RCKW alone or bound to DARPin E11. H and I , plots of Euler angle distribution generated in cryoSPARC ( left ) or Relion ( right ) for LRRK2 RCKW alone ( top ) or bound to DARPin E11 ( bottom ). Addition of E11 increased the number of orientations adopted by LRRK2 RCKW on the cryo-EM grids. DARPin, designed ankyrin-repeat protein; FSC, fourier shell correlation; LRRK2, leucine rich repeat kinase 2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of WT, full-length, untagged LRRK2 (pCDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene; RRID: Addgene_49543) or 1000 ng LRRK2, 500 ng GFP-Rab8A, and 500 ng of 8xHis-DARPin E11-3xFLAG using polyethylenimine.

Techniques: Cryo-EM Sample Prep, Binding Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2

doi: 10.1016/j.jbc.2024.107469

Figure Lengend Snippet: DARPin E11 does not affect the kinase activity of LRRK2 RCKW in vitro . A , in vitro assay measuring the level of Rab8A phosphorylation by LRRK2 RCKW . Rab8A and LRRK2 RCKW were incubated in the presence of ATP and kinase activity was monitored by measuring the levels of substrate (Rab8A) and product (pRab8A) by mass spectrometry. B , Kinase activity of LRRK2 RCKW in the presence of either DARPin E11 ( left ) or the LRRK2-specific kinase inhibitor GNE-9605 ( right ). The IC 50 was 38 nM ± 3 nM (SD). Plot points represent three technical replicates. DARPin, designed ankyrin-repeat protein; LRRK2, leucine rich repeat kinase 2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of WT, full-length, untagged LRRK2 (pCDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene; RRID: Addgene_49543) or 1000 ng LRRK2, 500 ng GFP-Rab8A, and 500 ng of 8xHis-DARPin E11-3xFLAG using polyethylenimine.

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Incubation, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2

doi: 10.1016/j.jbc.2024.107469

Figure Lengend Snippet: DARP in E11 disrupts LRRK2 FL filament formation and decreases Rab8 phosphorylation in cells . A , modeling of E11 bound to the autoinhibited conformation of LRRK2 FL shows no steric clashes between E11 and LRRK2. B , the binding site of DARPin E11 on the WD40 domain overlaps with the WD40:WD40 dimerization interface that is involved in the formation of microtubule associated LRRK2 filaments. C , Rab8 phosphorylation in 293T cells overexpressing LRRK2 FL and GFP-Rab8, with or without DARPin E11-3xFLAG. 293T cells were transiently cotransfected with LRRK2 FL and GFP-Rab8 or LRRK2 FL , GFP-Rab8, and DARPin E11-3xFLAG for 48 h. Cells transfected only with LRRK2 FL and GFP-Rab8 were treated with DMSO or 2 μM MLi-2 for 1 h. Cells were lysed and immunoblotted for phospho-Rab8 (pT72), total GFP-Rab8, total LRRK2, DARPin E11-3xFLAG, and GAPDH. MW; molecular weight marker. D , quantification from five Western blots plotting the ratio of GFP-pRab8 to total GFP-Rab8. Plot points represent four technical replicates. Statistics were generated in GraphPad using a one-way ANOVA analysis with a Tukey’s multiple comparison of means. ∗∗∗∗ p <0.0001 DMSO and MLi-2, ∗∗ p = 0.0013 DMSO and DARPin E11, ∗∗ p = 0.0012 MLi-2 and DARPin E11. E and F , representative images of 293T cells expressing either GFP-LRRK2 FL ( E ) or GFP-LRRK2 FL and DARPin E11 ( F ), treated with DMSO or MLi-2 for 2 h. G , quantification of the percent cells (mean ± sd) with GFP-LRRK2 FL filaments in the presence or absence or DARPin E11. Each data point on the graph represents a technical replicate. Three or four independent replicates were done per condition, with each replicate containing 48 to 140 cells. Statistics were generated using an one Way ANOVA with a Tuckey’s multiple comparison of means. ∗∗∗∗ p < 0.0001. ns, not significant. The tabular data for this figure can also be accessed at https://doi.org/10.5281/zenodo.10530220 . DARPin, designed ankyrin-repeat protein; DMSO, dimethylsulfoxide; LRRK2, leucine rich repeat kinase 2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of WT, full-length, untagged LRRK2 (pCDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene; RRID: Addgene_49543) or 1000 ng LRRK2, 500 ng GFP-Rab8A, and 500 ng of 8xHis-DARPin E11-3xFLAG using polyethylenimine.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Molecular Weight, Marker, Western Blot, Generated, Comparison, Expressing