Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Taurine activates the AMPK/NRF2 pathway by promoting the formation of the LKB1-STRAD-MO25 kinase complex (A) Quantitative abundance of taurine binding with LKB1-STRAD-MO25 complex subunits was measured using an LC-MS/MS-based trace-level detection method, n = 3. (B-C) Analysis of the binding of Taurine to purified LKB1 and MO25 WT proteins using surface plasmon resonance assay (SPR). (D) Plots of RMSD estimate averaged over all 10 trials versus simulation time for taurine docked with LKB1-STRAD-MO25 complex were calculated using Binding-pose metadynamics. A docking pose with a low PoseScore and high PersScore suggests a stable and reliable binding mode. (E) 3D diagram showing the most stable binding mode of taurine and LKB1-STRAD-MO25 (IFD2). (F) The LKB1 (Glu165 Arg301) and MO25 (Arg194 Leu197) sites were mutated to Ala, and quantitative abundance of taurine binding with LKB1-STRAD-MO25 complex subunits was measured using an LC-MS/MS-based trace-level detection method, n = 3. (G) The difference in the binding ability of LKB1 to MO25 in WT-NPCs and LKB1&MO25 point mutant NPCs (Mut-NPCs) was detected by co-immunoprecipitation after A1TP-HX-EVs treatment. Statistical analysis was performed using a two‐tailed unpaired Student's t‐test. n = 3, ∗∗ P < 0.01. (H) WT-NPCs and Mut-NPCs were induced with TBHP, and then treated with A1TP-HX-EVs for 24 h. Cell lysates were immunoblotted with antibodies against IVDD markers and ferroptosis-related proteins. (I) Cell lysates were immunoblotted with antibodies against AMPK signaling pathway-related proteins. (J) Representative images of mitochondrial morphology of TBHP-induced WT-NPCs and Mut-NPCs using TEM after 24 h of treatment as indicated. n = 3. Scale bar, 500 nm. (K-L) Representative oxygen consumption traces of WT-NPCs and Mut-NPCs induced with TBHP and then treated with A1TP-HX-EVs for 24 h. Maximal respiration of NPCs were quantified. All data are expressed as the mean ± SD. One‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. n = 3, ∗ P < 0.05. ∗∗ P < 0.01. ns, not significant.

Article Snippet: The primary antibodies included LKB1 (sc-32245, Santa Cruz Biotechnology), MO25 (2716S, Cell Signaling Technology), and STRAD (sc-515635, Santa Cruz Biotechnology).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Purification, SPR Assay, Mutagenesis, Immunoprecipitation, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

doi: 10.1101/2023.10.05.561145

Figure Lengend Snippet: Intramolecular disulfide bonds form in the kinase domains of BRSK1 and 2. (a) Full length BRSK1 and 2 were affinity-purified from HEK-293T cells and subjected to LC-MS/MS analysis. LC-MS/MS spectrum mapping revealed disulfide bridges formation between C147 BRSK1 - C153 BRSK1 , C191 BRSK1 - C198 BRSK1 , C132 BRSK2 - C138 BRSK2 , and C176 BRSK2 - C183 BRSK2 . (b) Alphafold structures demonstrating the location of disulfide bonds within the kinase domains of BRSK1 and BRSK2. (c) Real time phosphorylation of fluorescent AMARA peptide by the kinase domains of BRSK1 and 2 (100 ng). BRSK1 29- 358 and BRSK2 14-341 were activated by incubation with LKB1 and assayed in the presence of absence of 1 mM DTT.

Article Snippet: Active recombinant LKB1/STRADα/MO25α was purchased from Merck.

Techniques: Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: bioRxiv

Article Title: Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

doi: 10.1101/2023.10.05.561145

Figure Lengend Snippet: Biochemical analysis of BRSK Cys-to Ala mutants. (a) Immunoblot of in vitro glutathionylation of BRSK kinase domains. (b) Immunoblot showing LKB1-dependent phosphorylation of BRSK kinase domain proteins. (c) Thermal denaturation curves of BRSK catalytic domain proteins in the presence or absence of 10 mM DTT. (d) Thermal denaturation curves of BRSK catalytic domain cysteine to alanine mutants. (e) Representative immunoblot of EGFP- Tau co-expressed with full length, StWT and Cys-to-Ala mutants of BRSK1 and BRSK2. Transiently transfected HEK-293T cells were treated with or without 10 mM H 2 O 2 for 10 mins.

Article Snippet: Active recombinant LKB1/STRADα/MO25α was purchased from Merck.

Techniques: Western Blot, In Vitro, Transfection

Journal: bioRxiv

Article Title: Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

doi: 10.1101/2023.10.05.561145

Figure Lengend Snippet: Cysteine residues within the kinase domain fine-tune BRSK activity. In vitro kinase assays (right panels) showing normalized rates of peptide phosphorylation by WT and Cys-to-Ala variants of (a) BRSK1 and (b) BRSK2. 100 ng of LKB1 activated BRSK kinase domain was assayed in the presence or absence of 1 mM DTT. The positions of mutated Cys residues are modelled on the kinase domain as coloured spheres (left panel). Real time in vitro assays using (c) 50 ng BRSK1 and (d) 20 ng BRSK2. LKB1-activated BRSK proteins were incubated on ice in the presence or absence of 250 µM DTT for 30 mins. Assays were initiated by the addition of ATP and fluorescent peptide substrate in the presence or absence of 1 mM H 2 O 2 . All data is mean and SD of 3 experiments.

Article Snippet: Active recombinant LKB1/STRADα/MO25α was purchased from Merck.

Techniques: Activity Assay, In Vitro, Incubation