Targeting mannosylation of nicastrin N-glycans attenuates γ-secretase activity and notch-dependent leukemia progression
Hua Jiang, Weixiang Bian, Yanjun Cao, Zhuo Zhang, Yijia Chen, Yue Sui, Hongqiang Qin, Xu Li
Hua Jiang, Weixiang Bian, Yanjun Cao, Zhuo Zhang, Yijia Chen, Yue Sui, Hongqiang Qin, Xu Li
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Research Article Cell biology Oncology

Targeting mannosylation of nicastrin N-glycans attenuates γ-secretase activity and notch-dependent leukemia progression

Abstract

γ-Secretase is a transmembrane protease complex that cleaves multiple type I transmembrane proteins, including amyloid precursor protein and neurogenic locus notch homolog protein (NOTCH). Although numerous γ-secretase inhibitors and modulators targeting Notch-dependent cancers have been developed in recent decades, their clinical translation has been hampered by low substrate specificity and on-target gut toxicity. Using a proteomics-based screening approach, we identified dedicator of cytokinesis protein 2 (DOCK2) as an interactor of the γ-secretase subunit nicastrin (NCSTN). We further demonstrate that DOCK2 regulates mannosylation of NCSTN N-glycans, which in turn modulates γ-secretase activity toward NOTCH receptors. Both genetic depletion of DOCK2 and pharmacological inhibition of NCSTN mannosylation with kifunensine attenuated Notch-dependent leukemia progression in vivo. Collectively, these findings uncover a regulatory mechanism underlying substrate-specific activation of γ-secretase and suggest a promising therapeutic strategy for Notch-related diseases.

Authors

Hua Jiang, Weixiang Bian, Yanjun Cao, Zhuo Zhang, Yijia Chen, Yue Sui, Hongqiang Qin, Xu Li

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Figure 5

DOCK2 regulates NCSTN intracellular trafficking via N-glycosylation.

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DOCK2 regulates NCSTN intracellular trafficking via N-glycosylation. (A)...
(A) NCSTN protein level in DOCK2-KO HEK293T was evaluated. (B) WT and DOCK2-KO HEK293T cells were treated with 50 μg/mL cycloheximide (CHX) for the indicated times. Protein stability was evaluated and quantified. (C) Quantification of membrane-localized NCSTN mean fluorescence intensity in intact WT and DOCK2-KO JURKAT cells. (D) Subcellular fractionation of WT and DOCK2-KO JURKAT cells. Lysates were immunoblotted. (E and F) Immunofluorescence of WT and DOCK2-KO HEK293T cells transfected with GFP-NCSTN. Scale bars: 10 μm. (G) WT and DOCK2-KO JURKAT cells were treated with 2-bromohexadecanoic acid (2-BP) (50 μM, 12 h) and subjected to Western blotting. (H) Cell lysates from WT and DOCK2-KO JURKAT cells were treated with the glycosylases EndoH or PNGF and subjected to Western blotting. (I) Lectin blots of NCSTN immunoprecipitated from HEK293T cells. (J) HEK293T cells were cotransfected with Myc-DOCK2 and cSFB-tagged NCSTN or its glycosite mutants. The cell lysates were analyzed by Western blotting. (K) Validation of NCSTN-KO HEK293T cells generated via CRISPR/Cas9. (L) WT or NCSTN-KO HEK293T cells transfected with WT-NCSTN or N45Q mutant were subjected to a GVP-UAS–based γ-secretase assay. (M) Structural profiling of N-linked glycans at the NCSTN N45 site by MS. (N) WT and DOCK2-KO JURKAT cells were treated with N-butyl-deoxynojirimycin (NB-DNJ; 1 mM) or kifunensine (10 μM) for 24 h. The cell lysates were immunoblotted with NCSTN. (O) WT and DOCK2-KO JURKAT cells were pretreated with MG132 (10 μM) for 6 h, then the cell lysates were subjected to co-IP analysis against NCSTN and immunoblotted with ubiquitin (Ub). (P) Schematic illustrating regulation of NCSTN N-glycosylation by DOCK2 and inhibition by kifunensine. Created in BioRender. Mean ± SEM; A–L and N, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA.