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. 2022 Mar 7;25(3):103912. doi: 10.1016/j.isci.2022.103912

Figure 4.

Figure 4

TELO2 requires the C-terminal α-helix integrity for its binding to IVM B1a

(A) TELO2 deletion mutants lacking α-helices in the C-terminal region.

(B) Glutathione S-transferase (GST)-fusion TELO2 deletion mutants Δ5 and Δ6 lost the binding affinity for IVM B1a. Lysates from E. coli expressing GST-fusion TELO2 mutants were subjected to the binding assay. The lysates and the bound fractions were analyzed through western blotting with an anti-GST antibody. Arrows indicate the bands corresponding to TELO2 mutants.

(C) Sites of point mutations in the C-terminal α-helix are indicated as triangles.

(D) Site-directed mutagenesis in the C-terminal helix of TELO2 inhibited the binding of TELO2 to IVM B1a. Binding affinities of the point mutants were analyzed by probing the lysates and bound fractions with an anti-GST antibody.

(E) Chemical structures of IVM B1a-SNIPERs (specific and nongenetic IAP-dependent protein erasers) 3 and 4.

(F) IVM B1a-SNIPER promotes the proteasomal degradation of TELO2.

(G) IVM B1a-SNIPERs 3 and 4 reduced endogenous TELO2. HEK293 cells were treated with IVM B1a-SNIPERs 3 or 4 for 18 h in 1% FBS/DMEM. Then, the cell lysates were probed with anti-TELO2 and anti-actin antibodies (the left panel). The band intensities were quantified, normalized to the actin levels, and indicated as values relative to the control (DMSO) (the right panel). Data are presented as the means ± SDs (n = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA with Tukey’s test. See also Figure S3.

(H) Interaction of IVM B1a with TELO2 depended on K749 in cells. HEK293 cells were transfected with vectors encoding FLAG-tagged WT or K749T TELO2 and treated with 1 or 10 μM IVM B1a-SNIPER 3 for 5 h in 1% FBS/DMEM. Cell lysates were probed with anti-FLAG and anti-actin antibodies (the left panel). The band intensities were quantified, normalized to the actin levels, and indicated as values relative to the control (DMSO; the right panels). Data are presented as the means ± SDs (n = 3 biological replicates). ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Tukey’s test. See also Figure S4.