Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: D 2-hydroxyglutarate Inhibits Calcineurin Phosphatase Activity to Abolish NF-AT Activation and Interleukin-2 Induction in Stimulated Lymphocytes

doi: 10.4049/jimmunol.2200050

Figure Lengend Snippet: D2HG ligates calcineurin and suppresses its phosphatase activity. (A) D2HG and FK506 inhibition of NF-AT function and IL-2 production are additive. Jurkat cells were pre-incubated with 10 or 15 mM D2HG, or not, in the presence or absence of 1 nM FK-506 to inhibit calcineurin enzymatic activity. Luciferase and IL-2 expression 16 h later we determined as in the preceding panel. Triplicate assay replicates, n=2. (B) Surface plasmon resonance of immobilized calcineurin in response to D2HG or L2HG. Recombinant calcineurin was immobilized on anti-calcineurin conjugated CM5 Cytiva sensor chips with surface plasmon resonance response units assessed over time in media containing the stated concentrations of D2HG or L2HG. Representative of two identical experiments. (C) D2HG suppresses calcineurin enzymatic activity. Jurkat cells were pre-treated with the stated concentrations of D2HG for 30 min before activation with PMA and A23187 for 15 min. These cells were lysed and phosphatase activity was assessed in the presence or absence of EGTA and/or calcineurin. K506-inhibitable, Ca++-dependent activity as a function of D2HG concentration in triplicate assays of triplicate biologic replicates. (D) Stereochemical inhibition of calcineurin activity by D2HG. Jurkat cells were pre-treated for 30 min with the stated concentrations of D2HG or L2HG and then activated by PMA and A23187 for 15 min before the cells were lysed and FK506-inhibitable, Ca++-dependent phosphatase activity was assessed as in the preceding panel. n=3 separate biologic experiments with triplicate experimental replicates.

Article Snippet: Affinity and binding kinetics of 2HG for calcineurin was quantified using Series S CM5 chip on a Biacore (Cytiva) S200 model. Purified calcineurin (from Abcam Calcineurin Phosphatase Assay kit ab139461) was diluted (1:4) in acetate buffer (10 mM sodium acetate, pH 4.5) and immobilized on the surface of the sensor chips.

Techniques: Activity Assay, Inhibition, Incubation, Luciferase, Expressing, SPR Assay, Recombinant, Activation Assay, Concentration Assay