Journal: Journal of Neuroscience Research

Article Title: Tubulin Polymerization Promoting Proteins: Functional Diversity With Implications in Neurological Disorders

doi: 10.1002/jnr.70044

Figure Lengend Snippet: TPPP interacting proteins.

Article Snippet: There was increased α‐Syn aggregation and cell death in oligodendroglial KG1C cell lines when TPPP was expressed, which was partly rescued by SIRT2, a tubulin deacetylase (Hasegawa et al. ).

Techniques: Histone Deacetylase Assay, Binding Assay, Translocation Assay, Membrane

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet: Wild‐type GlyRS tightly binds the SIRT2 to inhibit its activity, not GlyRS CMT2D . (a) Coimmunoprecipitation of endogenous sirt2 showing specifically interaction with endogenous GlyRS in NSC‐34 cells. (b) Representative immunoblotting of 3 independent experiments shows that SIRT2 interacts with wild‐type GlyRS, not GlyRS mutant in stably expressing FLAG‐tagged GlyRS NSC34 cells FLAG‐tagged GlyRS was stably expressed in NSC34 cells. Precipitated GlyRS‐FLAG (WT, G526R, E71G) was detected by anti‐FLAG antibody, and co‐IP endogenous SIRT2 was detected by anti‐SIRT2 as indicated. (c) Representative immunoblotting of 3 independent experiments shows that SIRT2 interacts with wild‐type GlyRS, not GlyRS mutants in NSC‐34 cells. The NSC‐34 cells were transfected with GlyRS‐FLAG (WT, G526R, E71G, P234KY, C157R, G240R, L129P). Coimmunoprecipitations were performed with anti‐FLAG M2 magnetic beads. The immunoblot analysis was performed with anti‐Sirt2 and anti‐FLAG. (d‐e) Effect of GlyRS(d) or GlyRS mutant (G526R, E71G) (e) on SIRT2 deacetylation activity. SIRT2 (1 µM) was incubated with purified GlyRS or GlyRS (G526R, E71G) (concentration measured as a monomer) at the indicated ratios. The deacetylase activities of recombinant human SIRT2 were measured by monitoring the fluorescence intensity (excitation at 360 nm and emission at 460 nm) using a substrate peptide with one end coupled to a fluorophore and the other end to a quencher. A reaction without NAD + was performed as a negative control. Data are presented as mean ± SD, n = 3 biological replicates per group, from three independent experiments

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques: Activity Assay, Western Blot, Mutagenesis, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Transfection, Magnetic Beads, Incubation, Purification, Concentration Assay, Histone Deacetylase Assay, Recombinant, Fluorescence, Negative Control

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet: Wild‐type GlyRS binds the SIRT2 to maintain the acetylated a‐tubulin. (a–c) The recombinant SIRT2(1µmol) and recombinant GlyRSWT (a), GlyRSG526R (b), or GlyRSE71G (c) were incubated with NSC‐34 lysate at the indicated ratios(concentration measured as a monomer). The reaction products were detected by Western blotting for acetylated tubulin, a‐tubulin, and His‐tag. Data are presented as mean ± SD, n = 3 biological replicates per group, from three independent experiments. (d) Representative immunoblotting of 3 independent experiments shows that both knockdowns of GlyRS and overexpression of SIRT2 result in decreased tubulin acetylation. HEK293 cells were transfected with GlyRS siRNA or control siRNA 24 h later, and SIRT2‐Myc was overexpressed in cells transfected with control siRNA. After an additional 48 h, cells were harvested and acetylation of tubulin was detected by Western blot. (e) Western blot analysis detecting the level of α‐tubulin acetylation in NSC‐34 cells transfected with wild‐type GlyRS and GlyRS mutants

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques: Recombinant, Incubation, Concentration Assay, Western Blot, Over Expression, Transfection, Control

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet: The catalytic domain of GlyRS tightly binds the SIRT2. (a, b) Mapping study to identify the SIRT2 binding sites on GlyRS. FLAG‐tagged full‐length GlyRS or its truncated fragments was co‐transfected with HA‐tagged SIRT2 into HEK 293 cells. GlyRS proteins were immunoprecipitated with anti‐FLAG antibody, and the GlyRS ‐bound SIRT2 proteins were detected by Western blot using anti‐HA antibody. (c‐d) Mapping study to identify the GlyRS binding sites on SIRT2. HA‐tagged full‐length SIRT2 or its truncated fragments was co‐transfected with FLAG‐tagged GlyRS into HEK 293 cells. SIRT2 proteins were immunoprecipitated with anti‐HA antibody, and the SIRT2‐bound GlyRS proteins were detected by Western blot using anti‐FLAG antibody. (e) Structure of human GlyRS (PDB: 2ZT5 ) with the opened surface (red) caused by G526R and E71G mutations. (f) Electrostatic potential maps of WT GlyRS or GlyRS with E71G mutation. All images generated with PyMol

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Generated

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet: SIRT2 knockdown rescues CMT phenotype and life span in GlyRSG526R flies. (a) Bar graph displaying average climbing time to reach the target in a negative geotaxis assay of female flies in motor neurons (OK371‐GAL4). WT (gray), SIRT2 RNAi in GlyRSG526R (green), and GlyRSG526R (pink). N > 100. Error bars represent SEM. **** p < 0.0001. (b) 100 µM AGK2 was fed from 12‐h flies to 3‐ to 18‐day‐old flies; then, motor performance was detected and the bar graph was displayed average climbing time. AGK2‐fed GlyRSG526R (blue), SIRT2 RNAi in GlyRSG526R (green), and GlyRSG526R (pink). 4% DMSO was fed in all lines. N > 100. Error bars represent SEM. **** p < 0.0001. (c) SIRT2 knockdown restores NMJ in GlyRSG526R mutants. NMJs of third instar larvae expressing GlyRS in motor neurons (OK371‐GAL4) were visualized by staining for the postsynaptic marker disks large 1 (dlg1). Results indicate the NMJ on muscle 4, which is missing in GlyRSG526R flies and is rescued in SIRT2 knockdown GlyRSG526R flies. Scale bar, 50 mm. (d) Quantification of the percentage of animals with muscle 24 innervated; c2‐Test; *** p < 1 × 10 −6 ; N = 25. (e) Representative immunoblotting of 3 independent experiments shows that acetylated α‐tubulin in the lysate of different fly lines. (f) UAS‐SIRT2‐RNAi lines (BL31613) were bought from Tsinghua Fly Center and crossed with tub‐Gal4 as the control (UAS‐SIRT2 RNAi/+) for the longevity assay. Kaplan–Meier survival curves displaying the lifespan of male flies from the adult stage onwards. GlyRSG526R flies have a shorter median life span than GlyRSWT flies and Tubts control flies ( p < 0.0001, log‐rank test). N > 200 (g) SIRT2 knockdown or inhibitor extends the median life span of GlyRSG526R ( p < 0.0001, log‐rank test). N > 200

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques: Knockdown, Expressing, Staining, Marker, Western Blot, Control

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet: Schematics of targeting SIRT2 as a critical node between acetylated tubulin and CMT neuropathy

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques:

Journal: Aging Cell

Article Title: SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

doi: 10.1111/acel.13391

Figure Lengend Snippet:

Article Snippet: Compared with HDAC6, SIRT2 may function as a deacetylase of α‐tubulin in particular conditions, such as in the mitotic spindle (Nagai et al., ) or during the inflammasome activation in macrophages (Misawa et al., ).

Techniques: