Citation: Xian Yi, Zhou Mengxuan, Han Shangchen, Yang Rong, Wang Yi. A FRET biosensor reveals free zinc deficiency in diabetic betacell vesicles[J]. Chinese Chemical Letters, ;2020, 31(2): 468-472. doi: 10.1016/j.cclet.2019.08.012 shu

A FRET biosensor reveals free zinc deficiency in diabetic betacell vesicles

Xian Yi ^b,1, ,
Zhou Mengxuan ^c,1 ,
Han Shangchen ^b ,
Yang Rong ^b ,
Wang Yi ^a,*,

a.
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
b.
Peking-Tsinghua Center for Life Sciences, PKU-IDG/McGovern Institute for Brain Research, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
c.
State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China

xianyistevenx@outlook.com

wangyideyouxiang@sohu.com

Received Date: 27 May 2019
Revised Date: 8 August 2019
Accepted Date: 9 August 2019
Available Online: 12 August 2019

Figures(4)

The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement. However, common small-molecule intensity-based probes and most available genetically encoded Förster resonance energy transfer (FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles. Therefore, we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72, referred to as ZnT72R, which has an approximate K_d that varies from 6.07±0.28 μmol/L to 7.84±0.42 μmol/L in vitro and a cytosol-calibrated K_d of approximately 55.56±4.59 μmol/L in HEK293 T cells. To pinpoint the free zinc concentration of insulin-storing vesicles, we initially targeted ZnT72R to beta-cell vesicles by fusing them to NPY (neuropeptide Y). Following NPY-ZnT72R labeling, the FRET intensity ratios of vesicles were quantified. We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice (28.30±1.33 μmol/L) was significantly lower than that of control mice (41.46±3.53 μmol/L).
- Zinc,
- FRET,
- Insulin,
- Biosensor,
- Beta cell,
- Vesicle,
- Diabetes
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